siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAI, and stabilized forms thereof

ABSTRACT

Modification formats having modified nucleotides are provided for siRNA. Short interfering RNA having modification formats and modified nucleotides provided herein reduce off-target effects in RNA interference of endogenous genes. Further modification formatted siRNAs are demonstrated to be stabilized to nuclease-rich environments. Unexpectedly, increasing or maintaining strand bias, while necessary to maintain potency for endogenous RNA interference, is not sufficient for reducing off-target effects in cell biology assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of, and claims priorityto, U.S. patent application Ser. No. 13/975,136 filed Aug. 23, 2013,which application claims priority to U.S. patent application Ser. No.13/782,441 filed Mar. 1, 2013, which application claims priority to U.S.patent application Ser. No. 12/727,336 filed Mar. 19, 2010 (now U.S.Pat. No. 8,524,681, Sep. 3, 2013), which application claims priority toInternational Patent Application PCT/US2008/076675, having aninternational filing date of Sep. 17, 2008, which application claims thebenefit of U.S. Provisional Patent Application No. 60/973,548, filedSep. 19, 2007. Each application is incorporated by reference herein inits entirety.

FIELD

The present teachings generally relate to compositions, methods, andkits for reducing sequence-independent off-target phenotypic effects inRNA interference.

INTRODUCTION

Short interfering RNA (siRNA) potently induces cleavage of complementarymRNA rendering the mRNA nonfunctional with consequent loss of functionalprotein in a cell. The mechanism by which these molecules work has beenpartially characterized. Briefly, one of the two RNA strands of thesiRNA is incorporated into an enzyme complex termed RISC, which complexis then able to bind to and cleave a mRNA containing a complementarysequence thereby eliminating the mature mRNA from translation intoprotein. Each of the two strands of a siRNA can be incorporated into theRISC complex. However, the strand that has the weaker basepair at its5′-end is preferred for incorporation. Therefore, any siRNA will lead tothe production of a mixture of activated RISC complexes that can cleaveboth the intended target RNA as well as non-targeted RNA. In biologicalapplications, it is desirable that the vast majority, preferably all, ofthe activated RISC complexes contain the siRNA strand that iscomplementary to the desired target. The present teachings providemethods, compositions and kits for not only reducing mRNA cleavage bythe non-desired siRNA strand but also for reducing off-target effectsfor interference of endogenous genes.

SUMMARY

Unexpectedly, increasing or maintaining strand bias, while necessary tomaintain potency for endogenous RNA interference, is not sufficient forreducing off-target effects in cell biology assays. The ability toreduce or minimize off-target effects while maintaining potency wasstudied herein under conditions where the target mRNA was providedexogenously as well as where the target mRNA was an endogenous mRNA,under conditions where different types of modified nucleotides wereintroduced into siRNAs, and under conditions where differentmodification formats were introduced into siRNAs. Further, multiplesiRNA modification formats were studied for each of a number of targetRNAs. Therefore, modified nucleotides and modification formats thatreduce or minimize off-target events and that maintain potency of ahighly potent siRNA as observed at the phenotype and cell biology levelare applicable to any siRNA sequence and are not dependent on thesequence of the bases of a siRNA. That is, the modification formatsprovided herein are sequence-independent modifications.

In some embodiments, a chemically synthesized passenger (sense)oligonucleotide is provided, the oligonucleotide having a length of 15to 30 nucleotides and comprising one of sequence-independentmodification format (1), sequence-independent modification format (2),and sequence-independent modification format (3) as follows:

-   -   (1) 5′ N_(p)-m-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein p is 0 or 1;        x is 7, 8, 9, 10, or 11; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus (p+x+r+4);    -   (2) 5′ N_(p)-m-m-N_(y)-m-N_(z)-m-N_(q)-n_(r) 3′, wherein p is 0        or 1; y is 7, 8, or 9 and z is 1; or y is 7 and z is 2 or 3; r        is 0, 1, or 2; and q is an integer representing the length of        the passenger strand minus (p+y+z+r+4);    -   (3) 5′ m-N₁-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein x is 8, 9, or        10; r is 0, 1, or 2; and q is an integer representing the length        of the passenger strand minus (x+r+5);        wherein each m is independently a bicyclo nucleotide or a        tricyclo nucleotide; each n is independently a deoxynucleotide,        modified nucleotide, or ribonucleotide; when m is a bicyclo        nucleotide, each N is independently a nucleotide other than a        bicyclo nucleotide, and when m is a tricyclo nucleotide, each N        is independently a nucleotide other than a tricyclo nucleotide.

In some embodiments, a chemically synthesized short interfering RNA isprovided that comprises the passenger (sense) oligonucleotide describedabove and a guide (antisense) oligonucleotide having 15 to 30nucleotides, a region of continuous complementarity to the passenger(sense) oligonucleotide of at least 12 nucleotides; the guide strandfurther having complementarity to at least a portion of a target mRNA.

In some embodiments, a short interfering RNA is provided that comprisesa passenger (sense) oligonucleotide having a length of 17 to 30nucleotides and comprising one of sequence-independent modificationformat (4), format (5), and format (6) and a guide (antisense)oligonucleotide having 17 to 30 nucleotides, a region of continuouscomplementarity to the passenger (sense) oligonucleotide of at least 12nucleotides; the guide strand further having complementarity to at leasta portion of target mRNA,

-   -   (4) 5′ m_(p)-N_(x)-m-m-N_(q)-n_(r) 3′ wherein when p is 0, x is        12; when p is 1, x is 11; when p is 2, x is 10; when p is 3, x        is 9, when p is 4, x is 8; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus (p+x+r+2);    -   (5) 5′ m-m-N_(x)-m-N_(q)-n_(r) 3′ wherein x is 11; r is 0, 1, or        2; and q is an integer representing the length of the passenger        strand minus (x+r+3);    -   (6) 5′ m_(p)-N_(y)-m-N_(z)-m-N_(q)-n_(r) 3′, wherein p is 3; y        is 9 and z is 1; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus        (p+y+z+r+2);        wherein each m is independently a bicyclonucleotide, a        tricyclonucleotide, or a 2′-modified nucleotide; each n is        independently a deoxynucleotide, modified nucleotide, or        ribonucleotide and is an overhanging nucleotide; when m is a        bicyclonucleotide, each N is independently a nucleotide other        than a bicyclonucleotide, when m is a tricyclonucleotide, each N        is independently a nucleotide other than a tricyclonucleotide,        and when m is a 2′-modified nucleotide, each N is independently        a nucleotide other than a 2′-modified nucleotide.

In an embodiment of the short interfering RNA, the passenger (sense)oligonucleotide has sequence independent modification format (4), p is2, x is 10, r is 2, each n is an overhanging nucleotide, and each n isindependently a modified nucleotide; the guide (antisense)oligonucleotide comprises two 3′-overhanging modified nucleotides; andeach modified nucleotide is independently a bicyclonucleotide, atricyclonucleotide, or a 2′-modified nucleotide. In one embodiment, atleast one of the 3′ antepenultimate and the 3′ preantepenultimatepositions of the passenger oligonucleotide is a modified nucleotide. The3′ antepenultimate position is the third from the last position, i.e.,the position prior to the penultimate position, e.g., as for Format DH47of FIG. 1H. The 3′ preantepenultimate position is the fourth from thelast position, i.e., the position prior to the antepenultimate position,e.g., as for Format DH29 of FIG. 1H. Such siRNAs are particularly stableto nucleases such as in the presence of serum or plasma, for example.

In some embodiments of the short interfering RNA, when the length is 17nucleotides, at least one of positions 2 and 3 of the guideoligonucleotide is a modified nucleotide; when the length is 18nucleotides, at least one of positions 2, 3 and 4 of the guideoligonucleotide is a modified nucleotide; when the length is 19nucleotides, at least one of positions 2, 3, 4 and 5 of the guideoligonucleotide is a modified nucleotide; when the length is 20nucleotides, at least one of positions 2, 3, 4, 5 and 6 of the guideoligonucleotide is a modified nucleotide; and when the length is 21-30nucleotides, at least one of positions 2, 3, 4, 5, 6 and 7 of the guideoligonucleotide is a modified nucleotide. Formats DH1, and FormatsDH39-DH43 of FIG. 1K provide examples of siRNA modification formatshaving a length of 21 nucleotides and having at least one of positions2, 3, 4, 5, 6 and 7 of the guide oligonucleotide as a modifiednucleotide. In some embodiments, only one of positions 2, 3, 4, 5, 6 and7 (depending upon length as cited above) of the guide oligonucleotide isa modified nucleotide. Such siRNAs are also particularly stable tonucleases such as in the presence of serum, for example.

A method of reducing or minimizing off-target events for inhibition ofexpression of a target gene by RNA interference is provided thatcomprises contacting a cell containing the target gene with thechemically synthesized short interfering RNA described above whereineach m is independently a bicyclonucleotide or a tricyclonucleotide inan amount sufficient to reduce off-target events while maintainingpotency. The phrase “while maintaining potency,” is used herein in thecontext of use of a sufficient amount of siRNA to reduce off-targetevents while maintaining potency. In this context, the phrase refers tothe tolerance for reducing knockdown activity of the siRNA whilereducing off-target events and is dictated by whether the reducedknockdown activity elicits the gene associated phenotype. In general,“while maintaining potency” means that a reduction of knockdown to 80%of normal knockdown levels is acceptable. However, the tolerance forreduced knockdown of a specific gene can range from 50% to 95% of normalknockdown.

In further embodiments, a method of reducing or minimizing cleavage by apassenger strand in inhibition of expression of an exogenously providedtarget gene by RNA interference is provided, which method comprisescontacting a cell containing the target gene with a chemicallysynthesized short interfering RNA in an amount sufficient to reduce orminimize cleavage by a passenger strand while maintaining potency of aguide strand, the passenger strand of the short interfering RNAcomprising one of sequence-independent modification formats (1), (2),(3), (4), (5), or (6) wherein each m is independently a 2′-modifiednucleotide; each n is independently a deoxynucleotide, modifiednucleotide, or ribonucleotide; and each N is independently an unmodifiednucleotide. The reduction in cleavage is in comparison to that ofunmodified siRNA.

In some embodiments, the contacting is in vitro contacting of a cellculture containing the cell, or a tissue containing the cell with thechemically synthesized short interfering RNA. In further embodiments,the contacting is ex vivo contacting of a tissue containing the cell, abodily fluid containing the cell, or an organ containing the cell withthe chemically synthesized short interfering RNA. In yet otherembodiments, the contacting is in vivo contacting of an organ or ananimal containing the cell with the chemically synthesized shortinterfering RNA. In some embodiments, the contacting is in vivointravascular administration of a short interfering RNA havingmodification formats that confer nuclease stability thereto to a subjectin need thereof. As demonstrated by the data of Example 8, a greateramount of the stabilized short interfering RNA is present in vivo postadministration when compared to an amount present in vivo of controlshort interfering RNA lacking modified nucleotides at the same postadministration time point.

RNA interference assays provided by embodiments herein and carried outon exogenously provided genes demonstrate that a number of types ofmodified nucleotides in various modification formats provide forenhanced strand bias while maintaining potency. See data for Formats H,K, and O using bicyclo nucleotides of FIG. 4A and FIG. 4B and data forFormats H, K, and O using 2′-O-methyl nucleotides of FIG. 6A and FIG.6B.

Unexpectedly, those modified nucleotides and modification formatsdiffered in ability to reduce or minimize off-target events in RNAinterference of endogenous genes. A microarray analysis was carried outto determine the number of differentially expressed genes which is ameasure of off-target effects due to introduced siRNA. A differentiallyexpressed gene was defined as a gene having at least a 2-fold change dueto RNA interference with a p value of <0.001. LNA® nucleotide-modifiedFormat H siRNA produced 38% to 68% fewer differentially expressed genesas compared to the number of genes differentially expressed due tounmodified Format A siRNA having the same siRNA sequence. By comparison,2′-O-methyl modified Format H siRNA produced 0% to 22% fewerdifferentially expressed genes when compared to unmodified Format AsiRNA. Therefore, the LNA® nucleotide-modified siRNA is more effectiveat reducing off-target effects than 2′-O-methyl-modified siRNA asdetermined by global gene profiling analysis. See FIG. 9A-FIG. 9C.

These unexpected findings were confirmed using cell biology studies asillustrated by Example 6. When a collection of results was analyzed forsiRNA interference of endogenous genes, it was evident that the2′-O-methyl modified nucleotides in various modification formats asshown in FIG. 11C did not remove the “off-target” phenotypes to astatistically significant degree as compared to the unmodified format.These results are in contrast to those of FIG. 10E, which demonstrates astatistically significant ability of siRNAs having Format H to removeoff-target effects as evidenced by the lower apoptotic signal comparedto the unmodified Format A.

In some embodiments, the chemically synthesized short interfering RNA isfurther associated with a cell-targeting ligand as further describedbelow.

Compositions comprising the passenger oligonucleotide, the guideoligonucleotide, or the chemically synthesized short interfering RNA anda biologically acceptable carrier are contemplated as embodimentsherein. Further, kits that comprise the passenger oligonucleotide, theguide oligonucleotide, or chemically synthesized short interfering RNAand a transfection agent, for example, are contemplated as embodimentsherein. Short interfering RNAs having modification formats with modifiednucleotides of embodiments provide improved efficacy thus saving timedesigning and testing siRNAs, improved specificity thus saving time andmoney spent on spurious results, improved potency thus allowing for lessmaterial to be used to achieve the effect desired. Improved efficacy andspecificity also provide for use of fewer siRNAs per gene for individualgene and library screening studies.

In another embodiment, a method of reducing off-target effects on RNAinterference comprises obtaining siRNA having a passenger sense strandincluding a region homologous to a target transcribed RNA and having aguide antisense strand complementary to the sense strand, the siRNAhaving modification Format H, Q, V−2, Y, Y+1, JB1, JB2, JB3, JB4 or JB5,and contacting said siRNA with a cell. A measure of reduction ofoff-target effects is made in comparison to the off-target effects dueto an unmodified siRNA having the same sequence as themodification-formatted siRNA.

In a further embodiment, a method of increasing stability of siRNA in anuclease-rich environment comprises obtaining siRNA having a passengersense strand including a region homologous to a target transcribed RNA,having a guide antisense strand complementary to the sense strand, andhaving 3′-overhanging nucleotides, the siRNA having modification FormatDH47, DH29, DH1, DH39, DH40, DH41, DH42, or DH43, and contacting saidsiRNA with the nuclease rich environment. A measure of increasingstability of siRNA is made in comparison to the stability of siRNAhaving Format F, having the same type of modified nucleotide and havingthe same sequence.

Further embodiments include siRNAs having modification formats asprovided herein for use in RNA interference in in vitro, ex vivo, or invivo applications for inhibition of expression of a target gene. Whenexpression of a target gene is known to be associated with a disease,siRNAs herein are useful for screening for, diagnosis of or treatment ofsaid disease.

An unexpected aspect of the siRNA performance studies described hereinis the inability of reporter-based strandedness assays to predictperformance of a modification-formatted siRNA at the cellular phenotypiclevel. Differences in performance at the phenotypic level were observedusing microarray global gene profiling or cell-based assays as describedbelow.

These and other features of the present teachings will become moreapparent from the description herein.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A-FIG. 1K provide modification formats for siRNA studies. Numberslocated above a nucleotide indicate the position of the nucleotide inthe siRNA relative to the 5′ end of the format. m=a modified nucleotide,N=a nucleotide, or modified nucleotide having a modification other thanthe modification of m, n=nucleotide or modified nucleotide, P=aphosphate group.

FIG. 2A-FIG. 2B provide schematic diagrams depicting expression reportervectors used in various working examples. FIG. 2A shows a map of thepMIR-REPORT™ miRNA Expression Reporter Vector (Ambion/AppliedBiosystems) containing the firefly luciferase reporter gene. The codingregion of various genes of interest (GOI's) was inserted into theHindIII (nucleotide position 463) and SpeI (nucleotide position 525)restriction endonuclease sites within the multi-cloning site (MCS)located at the 3′ end of the luciferase gene. FIG. 2B shows a map of thepMIR-REPORT™ Beta-gal Control Vector (Ambion/Applied Biosystems)containing the β-galactosidase reporter gene. The control vector wasco-transfected with the pMIR-REPORT™ miRNA Expression Reporter Vectorcarrying the GOI and used as a transfection normalization control.

FIG. 3A-FIG. 3B provide schematic diagrams depicting the directionalrelationship between mRNA of a cloned GOI and the mRNA of the luciferasegene in the pMIR-REPORT™ miRNA expression vector. FIG. 3A provides theforward orientation, i.e., the 5′-end of the GOI mRNA is proximal to the3′-end of the fLuc mRNA. FIG. 3B provides the reverse orientation, i.e.,the 5′-end of the GOI mRNA is distal to the 3′-end of the fLuc mRNA.

FIG. 4A-FIG. 4E provide results of strand assays as described inExamples 2 and 3 for siRNAs having unmodified Format A and modificationformats as indicated.

FIG. 4A provides the amount of normalized reporter protein present aftercarrying out RNAi studies for siRNAs having modification formats asindicated. The dark bars represent knockdown activity by the guidestrand and the light bars represent knockdown activity by the passengerstrand. To display the distribution of the strandedness results of themodified siRNA strands, the data of FIG. 4B-FIG. 4E are provided in abox plot format, also referred to as a “box and whisker” plot. The darkhorizontal bar of a “box and whisker” plot represents the median valueof the dataset, the box represents the distribution of the data at thelower quartile and the upper quartile of the dataset, the dotted lines(whiskers) represent the smallest and largest values of the dataset, andthe ovals represent outliers in the dataset for each modificationformat.

FIG. 4B provides the knockdown activity of the siRNA passenger strandmeasured by the following formula: Passenger strand activity P=(fLUCactivity remaining from reverse clone treated with test siRNA/βgalactivity)/(fLUC activity remaining from reverse clone treated with Negcontrol siRNA/βgal activity). Wilcoxon-test (paired) results comparingeach modified format to the unmodified format are as follows: Format E,0.1434; Format F, 0.1011; Format G, 0.0002052; Format H, 0.0001755;Format J, 0.0002052; Format K, 0.006516; Format M, 0.002246; Format O,0.0009626.

FIG. 4C provides the knockdown activity of the guide strand measured bythe following formula: Guide strand activity G=(fLUC activity remainingfrom forward clone treated with test siRNA/βgal activity)/(fLUC activityremaining from forward clone treated with Neg control siRNA/βgalactivity). Wilcoxon-test (paired) results comparing each modified formatto the unmodified format are as follows: Format E, 0.0001271; Format F,0.8774; Format G, 0.0006498; Format H, 0.8115; Format J, 0.000006557;Format K, 0.01051; Format M, 0.7898; Format O, 0.8774.

FIG. 4D provides the difference of fLUC activity between the passengerand the guide strands of the siRNAs of FIG. 4B and FIG. 4C. The activitydifference was calculated by subtracting the activity of the normalizedforward fLUC activity from the normalized reverse fLUC activity orDifference=P−G. Thus, if the passenger strand is less active (lessknockdown, i.e., higher luciferase activity) than the guide strand(greater knockdown, i.e., lower luciferase activity), the difference ofactivity value is expected to be greater than 0. Values less than 0indicate that the passenger strand is more active than the guide strand.Wilcoxon-test (paired) results comparing each modified format to theunmodified format are as follows: Format E, 0.00792; Format F, 0.06043;Format G, 0.9664; Format H, 0.0014; Format J, 0.008715; Format K,0.6634; Format M, 0.002516; Format O, 0.0006498.

FIG. 4E demonstrates the activity fold change calculated as(log₂(P)−log₂(G)) which provides a measure of the impact of themodification format on the guide strand as compared to the impact of theformat on the passenger strand for the siRNAs of FIG. 4D. Thus, thelarger the activity fold change, the greater the guide strand bias ofthe siRNA. Wilcoxon-test (paired) results comparing each modified formatto the unmodified format are as follows: Format E, 0.0002781; Format F,0.08937; Format G, 0.2076; Format H, 0.01944; Format J, 0.0003223;Format K, 0.8553; Format M, 0.002516; Format O, 0.003504.

FIG. 5A-FIG. 5C provide results as described in Example 3 for studies inwhich the modification formats were strand-switched as compared to thestudies of FIG. 4A-FIG. 4E. That is, for modification F, for example,the 1, 20, 21 modification of the passenger (sense) strand wasintroduced into the guide (antisense) strand and the 20, 21 modificationof the guide (antisense) strand was introduced into the passenger(sense) strand.

FIG. 5A provides the amount of normalized reporter protein present aftercarrying out RNAi studies for 12 different siRNAs having themodifications of the strands switched. The dark bars represent knockdownactivity by the guide strand and the light bars represent knockdownactivity by the passenger strand.

FIG. 5B provides the difference in activity between the passenger strandand guide strand for the siRNAs of FIG. 5A in a box plot format.Wilcoxon-test (paired) results comparing each modified format to theunmodified format are identical for all formats at 0.03125.

FIG. 5C provides the fold change of activity between the passengerstrand and the guide strand for the siRNAs of FIG. 5B. Wilcoxon-test(paired) results comparing each modified format to the unmodified formatare identical for all formats at 0.03125.

FIG. 6A-FIG. 6C provide results of strand assays as described in Example3 for studies in which modified nucleotides of modification formats were2′-O methylated.

FIG. 6A provides the amount of normalized reporter protein present aftercarrying out RNAi studies for 6 different siRNAs having the modificationformats as indicated. The dark bars represent knockdown activity by theguide strand and the light bars represent knockdown activity by thepassenger strand.

FIG. 6B provides, in a box plot format, the difference of fLUC activitybetween the passenger strand and the guide strand for the set of siRNAsof FIG. 6A plus an additional set of 18 different siRNAs as described inExample 3. Wilcoxon-test (paired) results comparing each modified formatto the unmodified format are as follows: Format H−1, 0.001918; Format H,0.00001574; Format H+1, 0.001091; Format V−2, 0.0002131; Format K,0.0003619; Format O, 0.406.

FIG. 6C provides the fold change of activity between the passengerstrand and the guide strand for the siRNAs of FIG. 6B. Wilcoxon-test(paired) results comparing each modified format to the unmodified formatare as follows: Format H−1, 0.02514; Format H, 0.00001574; Format H+1,0.007443; Format V−2, 0.0001464; Format K, 0.0003619; Format O, 0.5446.

FIG. 7A-FIG. 7B provide results of strand assays as described in Example3 for studies in which modified nucleotides of modification formats were2′, 5′-linked nucleotides.

FIG. 7A provides the difference in guide and passenger strand knockdownactivity for siRNAs having unmodified Format A and siRNAs havingModification Formats H−1, H, H+1, V−2, K and O. Wilcoxon-test (paired)results comparing each modified format to the unmodified format are asfollows: Format H−1, 0.3594; Format H, 0.07422; Format H+1, 0.5703;Format V−2, 0.4258; Format K, 0.2031; Format O, 0.01953.

FIG. 7B provides the fold change of knockdown activity between thepassenger strand and the guide strand for the siRNAs of FIG. 7A.Wilcoxon-test (paired) results comparing each modified format to theunmodified format are as follows: Format H−1, 0.3008; Format H, 0.01953;Format H+1, 0.2031; Format V−2, 0.4961; Format K, 0.1921; Format O,0.00909.

FIG. 8 provides knockdown activity data for six siRNAs for each of 8endogenous targets. Each of the 48 siRNAs have modification formats asindicated, which formats incorporated bicyclo-modified nucleotides asdescribed in Example 4. Wilcoxon-test (paired) results comparing eachmodified format to the unmodified format are as follows: Format E,0.000000008657; Format F, 0.4631; Format G, 0.000009698; Format H,0.5569; Format J, 0.00000000191; Format K, 0.00001336; Format M, 0.7269;Format O, 0.0593.

FIG. 9A-FIG. 9C provide Venn diagrams of the intersection of 2-folddifferentially expressed genes between unmodified, 2′-O-methyl- orbicyclo-modified siRNAs as determined by microarray analysis and asdescribed by Example 5. For each diagram, the lower left set (red if incolor) indicates the genes changed 2-fold or greater in the unmodifiedsiRNA treated samples, the upper set (blue if in color) indicates thegenes changed 2-fold or greater in the 2′-O-methyl Format H modifiedsiRNA treated samples, and the lower right set (green if in color)indicates the genes changed 2-fold or greater in the bicyclo modifiednucleotide (LNA®) Format H modified siRNA treated samples.

FIG. 10A-FIG. 10E provide data on the impact of LNA® modificationformats on on-target and off-target phenotypes in cell biology studies.The negative control (Neg) is a scrambled non-targeting siRNA for whichmitosis or apoptosis quantitation is normalized to a value of 1.0. SeeExample 6.

FIG. 10A provides a box plot of normalized mitotic cells resulting fromsilencing by siRNA having LNA® modification Formats E, F, G, H, J, K, Mand O for a set of gene targets, the knockdown of a subset of which isexpected to increase mitosis, a subset of which is expected to have noeffect on mitosis, and a subset of which is expected to decrease mitosisas described by Example 6. Wilcoxon-test (paired) results comparing eachmodified format to the unmodified siRNA format are as follows: Format E,0.805; Format F, 0.5483; Format G, 0.3215; Format H, 0.004561; Format J,0.000952; Format K, 0.01974; Format M, 0.1007; Format O, 0.9438.

FIG. 10B provides a box plot as for FIG. 10A for a subset of genes,knockdown of which is expected to decrease mitosis. Wilcoxon-test(paired) results comparing each modified format to the unmodified siRNAformat are as follows: Format E, 0.3247; Format F, 0.4683; Format G,0.1815; Format H, 0.03423; Format J, 0.01387; Format K, 0.09874; FormatM, 0.5509; Format O, 0.6705.

FIG. 10C provides a box plot of normalized apoptotic fragments resultingfrom silencing by siRNA having LNA® modification Formats E, F, G, H, J,K, M and O for a set of gene targets, the knockdown of a subset of whichis expected to increase apoptosis and a subset of which is expected tohave no effect on apoptosis as described in Example 6. Wilcoxon-test(paired) results comparing each modified format to the unmodified siRNAformat are as follows: Format E, 0.2641; Format F, 0.5366; Format G,0.0001888; Format H, 0.0000102; Format J, 0.00000263; Format K,0.000000553; Format M, 0.5366; Format O, 0.05753.

FIG. 10D provides a box plot as for FIG. 10C for a subset of genes,knockdown of which is expected to increase apoptosis as described inExample 6. Wilcoxon-test (paired) results comparing each modified formatto the unmodified siRNA format are as follows: Format E, 0.2188; FormatF, 0.4375; Format G, 0.3125; Format H, 0.2188; Format J, 0.03125; FormatK, 0.03125; Format M, 1; Format O, 0.2188.

FIG. 10E provides a box plot as for FIG. 10C for a subset of genes,knockdown of which is expected to have no effect on apoptosis asdescribed in Example 6. However, siRNAs that have empiricallydemonstrated apoptosis off-target effects were specifically chosen forstudy to determine if such siRNAs, when modification-formatted, wouldeliminate or reduce the off-target effects. That the siRNAs haveoff-target effects is evidenced by the box plot for unmodified Format Awhich has an increased median and greater distribution of data ascompared to the Neg control. Wilcoxon-test (paired) results comparingeach modified format to the unmodified siRNA format are as follows:Format E, 0.5303; Format F, 0.804; Format G, 0.00005488; Format H,0.00001347; Format J, 0.0001253; Format K, 0.00003023; Format M, 0.441;Format O, 0.1551.

FIG. 11A-FIG. 11F provide data on the impact of 2′-O-methylatedmodification formats on on-target and off-target phenotypes in cellbiology studies. The negative control (Neg) is a scrambled non-targetingsiRNA for which mitosis or apoptosis quantitation is normalized to avalue of 1.0. See Example 6.

FIG. 11A provides a box plot of normalized mitotic cells resulting fromsilencing by siRNA having unmodified Format A, and having 2′-O-methylmodification Formats H and K for a set of gene targets, the knockdown ofa subset of which is expected to increase mitosis, and a subset of whichis expected to have no effect on mitosis as described by Example 6.Wilcoxon-test (paired) results comparing each modified format to theunmodified siRNA format are as follows: Format H, 0.3529; Format K,0.3289.

FIG. 11B provides a box plot as for FIG. 11A for the subset of genes,knockdown of which is expected to increase mitosis. Wilcoxon-test(paired) results comparing each modified format to the unmodified siRNAformat are as follows: Format H, 0.3125; Format K, 0.5469.

FIG. 11C provides a box plot as for FIG. 11A for the subset of genes,knockdown of which is expected to have no effect on mitosis. However,siRNAs that have empirically demonstrated mitosis off-target effectswere specifically chosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has a greater distribution of data ascompared to the Neg control. Wilcoxon-test (paired) results comparingeach modified format to the unmodified siRNA format are as follows:Format H, 0.9102; Format K, 0.4961.

FIG. 11D provides a box plot of normalized apoptotic fragments resultingfrom silencing by siRNA having 2′-O-methyl modification Formats H and Kfor a set of gene targets, the knockdown of a subset of which isexpected to increase apoptosis and a subset of which is expected to haveno effect on apoptosis as described in Example 6. Wilcoxon-test (paired)results comparing each modified format to the unmodified siRNA formatare as follows: Format H, 0.3529; Format K, 0.3529.

FIG. 11E provides a box plot as for FIG. 11D for the subset of genes,knockdown of which is expected to increase apoptosis as described inExample 6. Wilcoxon-test (paired) results comparing each modified formatto the unmodified siRNA format are as follows: Format H, 0.3125; FormatK, 0.5469.

FIG. 11F provides a box plot as for FIG. 11D for the subset of genes,knockdown of which is expected to have no effect on apoptosis asdescribed by Example 6. However, siRNAs that have empiricallydemonstrated apoptosis off-target effects were specifically chosen forstudy to determine if such siRNAs, when modification-formatted, wouldeliminate or reduce the off-target effects. Wilcoxon-test (paired)results comparing each modified format to the unmodified siRNA formatare as follows: Format H, 0.9102; Format K, 0.7344.

FIG. 12A-1 provides the difference in activity between the passengerstrand and guide strand calculated as P−G with P and G defined as forFIG. 4B and FIG. 4C for the unmodified format A and for test formats H,H+1, H−1, H−2, V, V−1, V−2, K and Q where the modification is LNA®residues. Wilcoxon-test (paired) results comparing each modified formatto the unmodified format are as follows: Format H, 0.04199; Format H+1,0.123; Format H−1, 0.1748; Format H−2, 0.2402; Format V, 0.06738; FormatV−1, 0.05371; Format V−2, 0.5771; Format K, 0.4648; Format Q, 0.1016.

FIG. 12A-2 provides the fold change of activity between the passengerstrand and the guide strand for the siRNAs of FIG. 12A-1. Wilcoxon-test(paired) results comparing each modified format to the unmodified formatare as follows: Format H, 0.3652; Format H+1, 0.2783; Format H−1,0.2402; Format H−2, 0.4131; Format V, 0.2061; Format V−1, 0.1016; FormatV−2, 0.7002; Format K, 0.6377; Format Q, 0.7002.

FIG. 12B provides knockdown activity data for endogenous targets forsiRNAs having unmodified Format A and for LNA® modification Formats H,H−2, H−1, H+1, Q, K, V, V−1, and V−2 as described in Example 6.Wilcoxon-test (paired) results comparing each modified format to theunmodified format are as follows: Format H, 0.029598160; Format H−2,0.004296849; Format H−1, 0.000833165; Format H+1, 0.052779900; Format Q,0.011454170; Format K, 0.745852000; Format V, 0.005660834; Format V−1,0.998100800; Format V−2, 0.374661100.

FIG. 12C provides a box plot of normalized mitotic cells resulting fromsilencing by siRNA having unmodified Format A and for LNA® modificationFormats H, H+1, H−1, H−2, K, Q, V, V−1, and V−2, for a set of genetargets, the knockdown of which is expected to increase mitosis asdescribed by Example 6. The negative control (Neg) is a scramblednon-targeting siRNA for which mitosis quantitation is normalized to avalue of 1.0. Wilcoxon-test (paired) results comparing each modifiedformat to the unmodified siRNA format are as follows: Format H, 0.01563;Format H+1, 0.1953; Format H−1, 0.07813; Format H−2, 0.07813; Format V,0.05469; Format V−1, 0.1953; Format V−2, 1; Format K, 0.1953; Format Q,0.1953.

FIG. 12D provides a box plot of normalized apoptotic fragments resultingfrom silencing by siRNA having unmodified Format A and for LNA®modification Formats H, H+1, H−1, H−2, K, Q, V, V−1, and V−2, for a setof gene targets, the knockdown of which is expected to have no effect onapoptosis. The negative control (Neg) is a scrambled non-targeting siRNAfor which apoptosis quantitation is normalized to a value of 1.0.However, siRNAs that have empirically demonstrated apoptosis off-targeteffects were specifically chosen for study to determine if such siRNAs,when modification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has an increased median and greaterdistribution of data as compared to the Neg control. Wilcoxon-test(paired) statistical significance calculations for normalized apoptoticfragments as a % of control comparing the unmodified Format A control tothe respective modification format are: Format H, 0.04199; Format H+1,0.2061; Format H−1, 0.2783; Format H−2, 0.6377; Format V, 0.05371;Format V−1, 0.4648; Format V−2, 0.006836; Format K, 0.01855; Format Q,0.024.

FIG. 13A-1 provides the difference in activity between the passengerstrand and guide strand calculated as P−G with P and G defined as forFIG. 4B and FIG. 4C for siRNAs having unmodified Format A andmodification Formats H, H+1, H−1, V−2, K and O where the modification is2′-O-methyl modified nucleotides. Wilcoxon-test (paired) resultscomparing each modified format to the unmodified format are as follows:Format H, 0.00001574; Format H+1, 0.001091; Format H−1, 0.001918; FormatV−2, 0.0002131; Format K, 0.0003619; Format O, 0.406.

FIG. 13A-2 provides the fold change of activity between the passengerstrand and the guide strand for the siRNAs of FIG. 13A-1. Wilcoxon-test(paired) results comparing each modified format to the unmodified formatare as follows: Format H, 0.00001574; Format H+1, 0.007443; Format H−1,0.02514; Format V−2, 0.0001464; Format K, 0.0003619; Format O, 0.5446.

FIG. 13B provides knockdown activity data for endogenous targets forsiRNAs having unmodified Format A and modification Formats H, H+1, H−1,K, V−2, and O where the modification is 2′-O-methyl modifiednucleotides. Wilcoxon-test (paired) results comparing each modifiedformat to the unmodified format are as follows: Format H, 3.47E-06;Format H+1, 0.001957517; Format H−1, 8.79E-09; Format K, 0.005446013;Format V−2, 1.95E-09; and Format O, 0.9972392.

FIG. 13C provides a box plot of normalized mitotic cells resulting fromsilencing by siRNA having unmodified Format A and for modificationFormats H, H−1, K and V having 2′-O-methyl modified nucleotides for aset of gene targets, the knockdown of which is expected to increasemitosis as described by Example 6. Wilcoxon-test (paired) resultscomparing each modified format to the unmodified format are as follows:Format H, 0.1953; Format H−1, 0.6406; Format V, 0.1953; Format K,0.6406.

FIG. 13D provides a box plot of normalized apoptotic fragments resultingfrom silencing by siRNAs having unmodified Format A and for modificationFormats H, H−1, V and K having 2′-O-methyl modified nucleotides for aset of gene targets, the knockdown of which is expected to have noeffect on apoptosis as described by Example 6. However, siRNAs that haveempirically demonstrated apoptosis off-target effects were specificallychosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has greater distribution of data ascompared to the Neg control. Wilcoxon-test (paired) results comparingthe unmodified Format A to each modification format are: Format H,0.9102; Format H−1, 0.25; Format V, 0.6523; Format K, 0.7344.

FIG. 14A provides a box plot of mRNA knockdown activity for endogenoustargets for siRNAs having unmodified Format A and for LNA® modificationFormats H, M, W, W+1, W−1, Y, Y+1, and Y−1. Wilcoxon-test (paired)statistical significance calculations comparing the unmodified Format Ato each modification format are: Format H, 0.005302; Format M, 0.02475;Format W, 1; Format W+1, 0.8423; Format W−1, 0.5096; Format Y, 0.932;Format Y+1, 0.887; Format Y−1, 0.5891.

FIG. 14B provides a box plot of mRNA knockdown activity for endogenoustargets for siRNAs having unmodified Format A and for LNA® modificationFormats H, M, JB1, JB2, JB3, JB4, and JB5. Wilcoxon-test (paired)statistical significance calculations comparing the unmodified Format Ato each modification format are: Format H, 0.005302; Format M, 0.02475;Format JB1, 0.009613; Format JB2, 0.7259; Format JB3, 0.4213; FormatJB4, 0.04904; Format JB5, 0.972.

FIG. 15A-FIG. 15D provide data on the impact of siRNAs having unmodifiedFormat A and for LNA® modification Formats H, M, W, W+1, W−1, Y, Y+1,and Y−1 on on-target and off-target phenotypes in cell biology studies.See Example 6.

FIG. 15A provides a box plot of normalized mitotic cells as described inExample 6 for a set of gene targets, knockdown of which is expected todecrease mitosis as described by Example 6. The negative control (Neg)is a scrambled non-targeting siRNA for which mitosis quantitation isnormalized to a value of 1.0. Wilcoxon-test (paired) results comparingeach modified format to the unmodified siRNA format are as follows:Format H, 0.4961; Format M, 1; Format W, 0.01953; Format W+1, 0.5703;Format W−1, 0.07422; Format Y, 0.03906; Format Y+1, 0.4961; Format Y−1,0.09766.

FIG. 15B provides a box plot as for FIG. 15A for a set of gene targets,knockdown of which is not expected to affect mitosis. However, siRNAsthat have empirically demonstrated mitosis off-target effects werespecifically chosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has a median value of about 0.7 ascompared to the Neg control. Wilcoxon-test (paired) statisticalsignificance calculations comparing the unmodified Format A control toeach modification format are: Format H, 0.5; Format M, 0.75; Format W,0.25; Format W+1, 0.25; Format W−1, 0.25; Format Y, 0.25; Format Y+1,0.25; Format Y−1, 1.

FIG. 15C provides a box plot of normalized apoptotic fragments for a setof gene targets, knockdown of which is expected to increase apoptosis asdescribed in Example 6. Wilcoxon-test (paired) results comparing eachmodified format to the unmodified siRNA format are as follows: Format H,0.75; Format M, 0.25; Format W, 0.25; Format W−1, 1; Format W+1, 0.25;Format Y, 0.25; Format Y−1, 0.25; Format Y+1, 0.25.

FIG. 15D provides a box plot as for FIG. 15C for a subset of genetargets, knockdown of which is not expected to have an effect onapoptosis. However, siRNAs that have empirically demonstrated apoptosisoff-target effects were specifically chosen for study to determine ifsuch siRNAs, when modification-formatted, would eliminate or reduce theoff-target effects. That the siRNAs have off-target effects is evidencedby the box plot for unmodified Format A which has a median value ofgreater than 3.5 and greater distribution of data as compared to the Negcontrol. Wilcoxon-test (paired) results comparing each modified formatto the unmodified siRNA format are as follows: Format H, 0.01221; FormatM, 0.8501; Format W, 0.07715; Format W+1, 0.06396; Format W−1, 0.1763;Format Y, 0.03418; Format Y+1, 0.021; Format Y−1, 0.2036.

FIG. 16A-FIG. 16D provide data on the impact of siRNAs having unmodifiedFormat A and for LNA® modification Formats H, M, JB1, JB2, JB3, JB4 andJB5 on on-target and off-target phenotypes in cell biology studies. SeeExample 6.

FIG. 16A provides a box plot of normalized mitotic cells for a set ofgene targets, knockdown of which is expected to decrease mitosis.Wilcoxon-test (paired) results comparing each modified format to theunmodified siRNA format are as follows: Format H, 0.4961; Format M, 1;Format JB1, 0.3594; Format JB2, 0.6523; Format JB3, 0.02734; Format JB4,0.1641; Format JB5, 0.2031.

FIG. 16B provides a box plot as for FIG. 16A for a set of gene targets,knockdown of which is not expected to affect mitosis. However, siRNAsthat have empirically demonstrated mitosis off-target effects werespecifically chosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has a median value of about 0.65thereby displaying a decrease in mitosis as compared to the Neg control.Wilcoxon-test (paired) results comparing the unmodified Format A controlto each modification format are: Format H, 0.5; Format M, 0.75; FormatJB1, 0.25; Format JB2, 0.25; Format JB3, 0.75; Format JB4, 0.25; FormatJB5, 0.25.

FIG. 16C provides a box plot of normalized apoptotic fragments resultingfrom silencing for a set of gene targets, the knockdown of which isexpected to increase apoptosis as described in Example 6. Wilcoxon-test(paired) results comparing each modified format to the unmodified siRNAformat are as follows: Format H, 0.75; Format M, 0.25; Format JB1, 0.25;Format JB2, 0.25; Format JB3, 0.25; Format JB4, 0.5; Format JB5, 0.75.

FIG. 16D provides a box plot as for FIG. 16C for a set of gene targets,knockdown of which is not expected to have an effect on apoptosis.However, siRNAs that have empirically demonstrated apoptosis off-targeteffects were specifically chosen for study to determine if such siRNAs,when modification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has an increased median value ofgreater than 3 and greater distribution of data as compared to the Negcontrol treated samples. Wilcoxon-test (paired) results comparing eachmodified format to the unmodified siRNA format are as follows: Format H,0.01221; Format M, 0.8501; Format JB1, 0.0009766; Format JB2, 0.0004883;Format JB3, 0.0004883; Format JB4, 0.003418; Format JB5, 0.003418.

FIG. 17 provides an assay for serum stability as a function of time forunmodified siRNAs in 90% serum at 37° C. The percentage of the remainingfull length siRNA duplex is shown for 8 different siRNAs at 0, 5, 10,15, 30, 60, 120 minutes incubation.

FIG. 18A provides a box plot of % of siRNAs that remain full length whentreated with 90% serum under conditions described in Example 7. Data areprovided for the unmodified Format A siRNA, siRNA modification Format Fwhich format is cited as siLNA5 by Elmen et al. (Nucleic Acids Research33:1, 439-447, 2005) and is used herein as a reference control forstability, and siRNA having modification Formats H, DH21, DH20, DH3,DH30, DH35, DH6, DH34, and DH2 where the modifications are LNA®residues. Wilcoxon-test (paired) results comparing Format F to eachmodification format are: Format H, 0.007813; Format DH21, 0.007813;Format DH20, 0.007813; Format DH3, 0.007813; Format DH30, 0.007813;Format DH35, 0.01563; Format DH6, 0.007813; Format DH34, 0.02488; FormatDH2, 0.2049.

FIG. 18B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNAs in Formats A, F, H, DH21,DH20, DH3, DH30, DH35, DH6, DH34, DH2 as described by Example 7.Wilcoxon-test (paired) statistical significance calculations comparingthe unmodified Format A control to each modification format are: FormatF, 0.05469; Format H, 0.3828; Format DH21, 0.007813; Format DH20,0.007813; Format DH3, 0.007813; Format DH30, 0.007813; Format DH35,0.01563; Format DH6, 0.007813; Format DH34, 0.03906; Format DH2, 0.6406.

FIG. 19A provides a box plot of % of siRNAs that remain full length whentreated with 90% serum under conditions described in Example 7. Data areprovided for modification Formats F, DH2, DH19, DH4, DH31, DH27, andDH25 where the modifications are LNA® residues. Wilcoxon-test (paired)statistical significance calculations comparing Format F to eachmodification format are: Format DH2, 0.2049; Format DH19, 0.1953; FormatDH4, 0.02249; Format DH31, 0.01563; Format DH27, 0.1094; Format DH25,0.1094.

FIG. 19B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNA in Formats F, DH2, DH19,DH4, DH31, DH27, and DH25 as described by Example 7. Wilcoxon-test(paired) statistical significance calculations comparing the unmodifiedFormat A control to each modification format are: Format F, 0.05469;Format DH2, 0.6406; Format DH19, 0.01563; Format DH4, 0.007813; FormatDH31, 0.01563; Format DH27, 0.01563; Format DH25, 0.007813.

FIG. 20A provides a box plot of % of siRNAs that remain full length whentreated with serum under conditions described in Example 7. Data areprovided for modification Formats F, DH2, DH47, DH29, DH28 and DH18where the modifications are LNA® residues. Wilcoxon-test (paired)statistical significance calculations comparing Format F to eachmodification format are: Format DH2, 0.2049; Format DH47, 0.03906;Format DH29, 0.07813; Format DH28, 0.01415; Format DH18, 0.06836.

FIG. 20B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNA in Formats F, DH2, DH47,DH29, DH28 and DH18 as described by Example 7. Wilcoxon-test (paired)statistical significance calculations comparing the unmodified Format Acontrol to each modification format are: F, 0.05469; Format DH2, 0.6406;Format DH47, 0.3125; Format DH29, 0.02249; Format DH28, 0.01563; FormatDH18, 0.01563.)

FIG. 21A provides a box plot of % of siRNAs that remain full length whentreated with serum under conditions described in Example 7. Data areprovided for modification Formats F, DH36, DH2, DH9, DH46, DH33 and DH10where the modifications are LNA® residues. Wilcoxon-test (paired)statistical significance calculations comparing Format F to eachmodification format are: Format DH36, 0.1609; Format DH2, 0.2049; FormatDH9, 0.5534; Format DH46, 0.02071; Format DH33, 0.1508; Format DH10,0.05469.

FIG. 21B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNAs in Formats F, DH36, DH2,DH9, DH46, DH33 and DH10 as described by Example 7. Wilcoxon-test(paired) statistical significance calculations comparing unmodifiedFormat A control to each modification format are: Format F, 0.05469;Format DH36, 0.01563; Format DH2, 0.6406; Format DH9, 0.1484; FormatDH46, 0.1484; Format DH33, 0.007813; Format DH10, 0.007813.

FIG. 22A provides a box plot of % of siRNAs that remain full length whentreated with serum under conditions described in Example 7. Data areprovided for modification Formats F, DH2, DH7, DH23, DH1, DH48, DH49,DH44 and DH45 where the modifications are LNA® residues. Wilcoxon-test(paired) statistical significance calculations comparing Format F toeach modification format are: Format DH2, 0.2049; Format DH7, 0.3828;Format DH23, 0.03461; Format DH1, 0.1484; Format DH48, 0.6406; FormatDH49, 0.1410; Format DH44, 0.3125; Format DH45, 0.1052.

FIG. 22B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNAs in Formats F, DH2, DH7,DH23, DH1, DH48, DH49, DH44 and DH45 as described by Example 7.Wilcoxon-test (paired) statistical significance calculations comparingunmodified Format A to each modification format are: Format F, 0.05469;Format DH2, 0.6406; Format DH7, 0.05469; Format DH23, 0.007813; FormatDH1, 0.007813; Format DH48, 0.1953; Format DH49, 0.4609; Format DH44,0.007813; Format DH45, 0.01563.

FIG. 23A provides a box plot of % of siRNAs that remain full length whentreated with serum under conditions described in Example 7. Data areprovided for modification Formats F, DH2, DH38, DH1, DH39, DH40, DH41,DH42 and DH43 where the modifications are LNA® residues. Wilcoxon-test(paired) statistical significance calculations comparing Format F toeach modification format are: Format DH2, 0.2049; Format DH38, 0.04206;Format DH1, 0.1484; Format DH39, 0.07813; Format DH40, 0.2500; FormatDH41, 0.2620; Format DH42, 0.1094; Format DH43, 0.3621.

FIG. 23B provides a box plot of the fraction of mRNA knockdown, relativeto Neg control siRNA, by LNA® modified siRNAs in Formats F, DH2, DH38,DH1, DH39, DH40, DH41, DH42 and DH43 as described by Example 7.Wilcoxon-test (paired) statistical significance calculations comparingunmodified Format A to each modification format are: Format F, 0.05469;Format DH2, 0.6406; Format DH38, 0.007813; Format DH1, 0.007813; FormatDH39, 0.007813; Format DH40, 0.01563; Format DH41, 0.007813; FormatDH42, 0.007813; Format DH43, 0.02344.

FIG. 24 provides quantification of in vivo presence of stabilizedsiRNAs. The amount of siRNA present in livers from spiked controlanimals is compared to the amount of siRNA present in livers from testanimals injected with unmodified Format A siRNA and LNA®modification-formatted stabilized siRNAs having Format DH1 or FormatDH47. CT is cycle threshold.

DESCRIPTION OF VARIOUS EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. For example, “at least one” means thatmore than one can be present. Also, the use of “comprise”, “contain”,and “include”, or modifications of those root words, for example but notlimited to, “comprises”, “contained”, and “including”, are not intendedto be limiting and means “including the following elements but notexcluding others.” The term “consists essentially of,” or “consistingessentially of,” as used herein, excludes other elements from having anyessential significance to the combination. Use of “or” means “and/or”unless stated otherwise. The term “and/or” means that the terms beforeand after can be taken together or separately. For illustrationpurposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or“X and Y”.

Whenever a range of values is provided herein, the range is meant toinclude the starting value and the ending value and any value or valuerange there between unless otherwise specifically stated. For example,“from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35,0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39;and the like.

All literature and similar materials cited in this applicationincluding, but not limited to, patents, patent applications, articles,books, treatises, and internet web pages, regardless of the format ofsuch literature and similar materials, are expressly incorporated byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines oruses a term in such a way that it contradicts that term's definition inthis application, this application controls. While the present teachingsare described in conjunction with various embodiments, it is notintended that the present teachings be limited to such embodiments. Onthe contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.The term “surrogate” as used herein means a product that is indicativeof presence of another product. For example, an amplification product isa surrogate for a nucleic acid that has been amplified.

As used herein, “modification format,” for a siRNA means a pattern ofmodified nucleotides and “other-than modified” nucleotides asexemplified by the patterns or formats set forth by FIG. 1A-FIG. 1K.Modified nucleotides are described below. “Other-than modified” means anunmodified nucleotide or a modified nucleotide having a modificationother than the modification of the modified nucleotide in a particularformat.

As used herein, “sequence-independent modification,” means that modifiednucleotides and modification formats provided by embodiments may beapplied to any siRNA sequence without regard to the particular sequenceof that siRNA.

An “on-target” event, as used herein, means that the mRNA of a targetgene is impacted, i.e., knocked-down, by the siRNA designed to targetthat gene as evidenced by reduced expression, reduced levels of mRNA, orloss or gain of a particular phenotype. An “on-target” event can beverified via another method such as using a drug that is known to affectthe target, or such as rescuing a lost phenotype by introduction of thetarget mRNA from an ortholog, for example.

An “off-target event,” as used herein, means any event other than thedesired event in RNA interference. An example of an off-target event iswhere a mRNA of a gene that is not targeted is impacted, i.e.,knocked-down, by an siRNA. Such “off-target events” cannot be rescued bysupplying the target mRNA from an appropriately related ortholog.“Off-target events” may be due to interaction with another mRNA,endogenous RNA, a DNA, or a protein in a way that alters non-targetexpression. Such off-target events may be due to, for example, mismatchbase pairing between a nucleotide sequence incorporated into the RISCand a non-target mRNA, or non-specific interactions within the cellularmileau. Inappropriate RISC binding, inappropriate RISC-mediatedinteractions, and inappropriate RISC-mediated cleavage may contribute tooff-target events. Off-target events also may be due to cytotoxicresponses, an interferon response, a microRNA effect, or interactionwith non-coding RNA. The most common off-target event is likely theundesired incorporation of the passenger (sense) strand into the RISCcomplex. However, the off-target events that are affected by siRNAs arenot entirely predictable and tend to be non-specific. Off-target eventsgive rise to off-target effects and the terms are used interchangeablyherein.

The term “potency,” as used herein, is a measure of the concentration ofan individual or a pool of siRNA required to knock down its target mRNAto 50% of the starting mRNA level. Generally, potency is described interms of IC50, the concentration of siRNA required for half maximum(50%) mRNA inhibition. Potency is determined herein by transfecting arange of siRNA concentrations (e.g., at least six ranging from 100 nM to10 pm) and performing qRT-PCR analysis on the samples. The results areplotted and the IC50 is determined by extrapolating against theconcentration where 50% of mRNA knockdown is achieved. A curve-fittingprogram is used to determine the concentration of siRNA needed toachieve 50% knockdown of the mRNA target. As used herein, the term“potency” is used interchangeably with the term “knockdown activity”which is a measure of the amount of decrease of mRNA after exposure to atest siRNA as compared to the amount of mRNA present after exposure to anegative control siRNA.

The term “efficacy,” as used herein, is the percent of siRNA thatproduces a minimum threshold of mRNA knockdown. The term efficacy iscommonly applied to a collection of siRNAs or to the predictive power ofa siRNA design algorithm. Efficacy is measured by qRT-PCR orvector-based measurements of siRNA knockdown of a target mRNA, whichresults are expressed as the % of siRNAs that produce a minimumthreshold of knockdown, e.g., % of siRNA that knockdown at 70% orbetter.

The term “specificity,” as used herein, is a measure of the precisionwith which a siRNA impacts gene regulation at the mRNA level or proteinlevel, or the true phenotype exhibited by knockdown of the target genefunction. Specificity is measured by determining the number of genesthat are changed relative to a certain threshold as a result oftransfection of the siRNA, are not the intended target, and are notknown to be in the signal transduction pathway of the target gene. Theindustry standard threshold is a 2-fold change. In cell biology studies,highly specific siRNAs to a particular target result in overlappingphenotypes with little to no unexpected phenotypes. Poor specificitysiRNAs result in a phenotype due to off-target effects.

“Improved” potency refers to a siRNA having a lower IC50 or a higherpercentage knockdown at the same concentration than another siRNA,“improved” specificity refers to a siRNA that impacts fewer genes thananother siRNA, and “improved” efficacy refers to a group of siRNAsgenerated using an algorithm where a larger percentage of the siRNAgroup produces a minimum designated amount of knockdown as compared toanother siRNA group generated using a different algorithm.

For studies herein, test results are compared to control results, andall results are normalized to a nonsense siRNA designed to benon-targeting. The Wilcoxon test for paired samples is thenon-parametric equivalent of the paired samples t-test. The Wilcoxontest ranks the absolute values of the differences between the paireddata in sample 1 and sample 2 and calculates a statistic on the numberof negative and positive differences (differences are calculated assample 2−sample 1). The software used is the “R Package” available at nocharge from cran.org.

The term “nucleotide” generally refers to a phosphate ester of anucleoside, either as a monomer or within a dinucleotide,oligonucleotide or polynucleotide. A nucleoside generally is a purinebase or a pyrimidine base linked to the C-1′ carbon of a ribose (aribonucleoside) or of a deoxyribose (deoxyribonucleoside). Naturallyoccurring purine bases generally include adenine (A) and guanine (G).Naturally occurring pyrimidine bases generally include cytosine (C),uracil (U) and thymine (T). When the nucleoside base is a purine, theribose or deoxyribose is attached to the nucleobase at the 9-position ofthe purine, and when the nucleobase is a pyrimidine, the ribose ordeoxyribose is attached to the nucleobase at the 1-position of thepyrimidine. A ribonucleotide is a phosphate ester of a ribonucleosideand a deoxyribonucleotide is a phosphate ester of a deoxyribonucleoside.The term “nucleotide” is generic to both ribonucleotides anddeoxyribonucleotides. A dinucleotide generally has two nucleotidescovalently bonded via a 3′-5′ phosphodiester linkage. An oligonucleotidegenerally has more than two nucleotides and a polynucleotide generallyrefers to polymers of nucleotide monomers. Applicants have used theterms “nucleotide” and “nucleoside” interchangeably herein forconvenience although one of ordinary skill in the art readilyunderstands, from the context in which the terms appear, which term isapplicable.

Nucleotide monomers are linked by “internucleoside or internucleotidelinkages,” e.g., phosphodiester linkages where, as used herein, the term“phosphodiester linkage” refers to phosphodiester bonds or bondsincluding phosphate analogs thereof, including associated counterions,e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions are present. Furtherinternucleoside or internucleotide linkages are described below.Whenever an oligonucleotide is represented by a sequence of letters, itwill be understood that the nucleotides are in 5′ to 3′ order from leftto right unless otherwise noted or it is apparent to the skilled artisanfrom the context that the converse was intended. Descriptions of how tosynthesize oligonucleotides can be found, among other places, in U.S.Pat. Nos. 4,373,071; 4,401,796; 4,415,732; 4,458,066; 4,500,707;4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319; and 5,262,530.Oligonucleotides can be of any length.

“Base pairing,” as used herein, refers to standard Watson-Crick basepairing. Base pairings found commonly in double-stranded (duplex)nucleic acids are G:C, A:T, and A:U. Such base pairs are referred to ascomplementary base pairs and one base is complementary to its pairedbase. Nucleotide analogs as described infra are also capable of forminghydrogen bonds when paired with a complementary nucleotide or nucleotideanalog.

As used herein, the terms “complementary” or “complementarity” are usednot only in reference to base pairs but also in reference toanti-parallel strands of oligonucleotides related by the Watson-Crickbase-pairing rules, to nucleic acid sequences capable of base-pairingaccording to the standard complementary rules, or being capable ofhybridizing to a particular nucleic acid segment under relativelystringent conditions. For example, the sequence 5′-AGTTC-3′ iscomplementary to the sequence 5′-GAACT-3′. The terms “completelycomplementary” or “100% complementary” and the like refer tocomplementary sequences that have perfect pairing of bases between theanti-parallel strands (no mismatches in the polynucleotide duplex).Nucleic acid polymers can be complementary across only portions of theirentire sequences. The terms “partial complementarity,” “partiallycomplementary,” “incomplete complementarity” or “incompletelycomplementary” and the like refer to any alignment of bases betweenanti-parallel polynucleotide strands that is less than 100% perfect(e.g., there exists at least one mismatch in the polynucleotide duplex).Furthermore, two sequences are said to be complementary over a portionof their length if there exists one or more mismatches, gaps orinsertions in their alignment.

Furthermore, a “complement” of a target polynucleotide refers to apolynucleotide that can combine in an anti-parallel association with atleast a portion of the target polynucleotide. The anti-parallelassociation can be intramolecular, e.g., in the form of a hairpin loopwithin a nucleic acid molecule, or intermolecular, such as when two ormore single-stranded nucleic acid molecules hybridize with one another.The term “corresponding to” when in reference to nucleic acids, meansthat a particular sequence is sufficiently complementary to ananti-parallel sequence such that the two sequences will anneal and forma duplex under appropriate conditions.

As used herein, a “passenger (sense)” strand, region, or oligonucleotiderefers to a nucleotide sequence that has the same nucleotide sequence ofat least a portion of the sense strand of DNA or of the nucleotidesequence of at least a portion of mRNA. A “passenger (sense) strand”includes a sense region of a polynucleotide that forms a duplex with acomplementary guide (antisense) region of another polynucleotide. Ahairpin single-stranded oligonucleotide (shRNA) may also contain apassenger (sense) region and a guide (antisense) region within the samemolecule, the regions forming a duplex structure joined by a loopsequence or loop linker moiety as described further below. A hairpin canbe in either a left-handed orientation (i.e.,5′-antisense-loop-sense-3′) or a right-handed orientation (i.e.,5′-sense-loop-antisense-3′).

As used herein, a “siRNA” refers to a short interfering RNA duplex thatinduces gene silencing via a RNA interference (RNAi) pathway. Shortinterfering RNAs can vary in length, and can contain at most one or twomismatched base pairs between the antisense and sense regions, andbetween the antisense region and a target mRNA sequence. Each siRNA caninclude 15 to 30 base pairs, or 18 to 25 base pairs, or 19 to 22 basepairs or 21 base pairs or 19 base pairs. In some embodiments, siRNAshave, independently, 1, 2, 3, 4, or 5 unpaired overhanging nucleotideson the 5′ end, the 3′ end, or both the 5′ end and the 3′ end. In someembodiments, siRNAs have blunt ends. In addition, the term “siRNA”includes duplexes of two separate strands, as well as single strandsthat can form hairpin structures (a shRNA). A shRNA may have a loopsequence of nucleotides such as 4 to 30, or 6 to 20 or 7 to 15nucleotides or may comprise non-nucleotide moieties such as hydrocarbonlinking regions, or a combination thereof. A shRNA may comprisemismatches or bulges.

In some embodiments, the present teachings comprise modified nucleotidesthat have enhanced affinity for base pairing as compared to non-modifiednucleotides or that contribute enhanced affinity to the nucleotidesequence of which it is part. Modified nucleotides, as contemplatedherein, shift the conformational equilibrium of an RNA toward thenorthern (C3′-endo) conformation consistent with the A-form geometry ofRNA duplexes. DNA:RNA duplexes can also be stabilized thereby. In someembodiments, enhanced affinity for base pairing is achieved byconstructing a passenger (sense) oligonucleotide having abicyclonucleotide, tricyclonucleotide or a 2′-modified nucleotide.

In some embodiments, modified nucleotides for siRNA include,independently, modified sugar portions, modified base portions, modifiedinternucleoside portions, or a combination of any of these modifiedportions.

In some embodiments, modified nucleotides include modified sugarportions including, but not limited to, 2′-halo where halo is chloro,fluoro, bromo or iodo; arabino or 2′-fluoro in an arabinose conformation(FANA); 2′-H, —SH, —NH₂, —CN, azide; or —OR, —R, —SR, —NHR, or —N(R)₂,wherein R is alkyl, alkenyl, alkynyl, alkoxy, oxyalkyl, alkoxyalkyl,alkylamine, where the alkyl portion is C1-C6.

Further 2′ modifications include 2′-O—(CH₂)₄NH₂;2′-O-anthraquinolylalkyl where alkyl comprises methyl or ethyl;2′-O—(CH₂)₂—OCH₃; 2′-O—(CH₂CH₂O)_(n)—CH₃ where n is 1-4; 2′-O—CH₂—CHR—Xwhere X═OH, F, CF₃ or OCH₃ and R═H, CH₃, CH₂OH or CH₂OCH₃; peptidenucleic acid monomers or derivatives thereof (PNA); 2′ modificationswhere the 2′-position is alkoxy, e.g., methoxy, ethoxy, allyloxy,isopropoxy, butoxy, isobutoxy and phenoxy, and the like.

In some embodiments, modified nucleotides include modified sugarportions or pseudosugars including, but not limited to,bicyclonucleotide structure (a), tricyclonucleotide structure (b), orbicyclonucleotide structure (c):

wherein R1 and R2 are independently O, S, CH₂, or NR where R is hydrogenor C₁₋₃-alkyl; R3 is CH₂, CH₂—O, CH₂—S, CH₂—CH₂, CH₂—CH₂—CH₂, CH═CH, orCH₂—NR where R is hydrogen or C₁₋₃-alkyl; R4 and R5 are independently aninternucleoside linkage, a terminal group, or a protecting group; atleast one of R4 and R5 is an internucleoside linkage; and B is anucleobase, nucleobase derivative, or nucleobase analog. In someembodiments, R4 and R5 are independently H, OH, phosphate, C₁₋₁₂-alkyl,C₁₋₁₂-alkylamine, C₁₋₁₂-alkenyl, C₁₋₁₂-alkynyl, C₁₋₁₂-cycloalkyl,C₁₋₁₂-aralkyl, aryl, acyl, silyl, an oligonucleotide, a nucleoside boundvia an internucleoside linkage, or a combination thereof; and at leastone of R4 and R5 is an oligonucleotide, or a nucleoside bound via aninternucleoside linkage. The 5′ ends may further be derivatized bysubstituent groups such as phosphate, C₁₋₁₂-alkyl, C₁₋₁₂-alkylamine,C₁₋₁₂-alkenyl, C₁₋₁₂-alkynyl, C₁₋₁₂-cycloalkyl, C₁₋₁₂-aralkyl, aryl,acyl, or silyl. In some embodiments, the 5′-ends are derivatized by C₁₂or C₆ substituted groups, or by phosphate, for example.

Bicyclonucleotides include those described above for structures (a) and(c). The bicyclo nucleotides of structure (a) are also termed lockednucleic acids or LNA® and include β-D, and α-L bicyclo nucleotides,bicyclo nucleotides such as xylo-locked nucleic acids (U.S. Pat. No.7,084,125), L-ribo-locked nucleic acids (U.S. Pat. No. 7,053,207), 1′-2′locked nucleic acids (U.S. Pat. Nos. 6,734,291 and 6,639,059), and 3′-5′locked nucleic acids (U.S. Pat. No. 6,083,482), for example.Bicyclonucleotides of structure (c) and synthesis of oligonucleotidescontaining such bicyclonucleotides are described in, e.g., Maier et al.(Nucleic Acids Research 2004, 32:12, 3642-3650) and Maderia et al.(Nucleic Acids Research 2007, 35:6, 1978-1991). Tricyclonucleotides ofstructure (b) and synthesis of oligonucleotides containing suchtricyclonucleotides are described in e.g., Ittig et al. (Nucleic AcidsResearch 2004, 32:1, 346-353).

In some embodiments, nucleotides include modified internucleoside linkerportions including, but not limited to, phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, thioformacetal (—S—CH₂—O—CH₂—),methylene(methylimino), dimethylhydrazino, phosphoryl linked morpholino,—CH₂—CO—NH—CH₂—, —CH₂—NH—CO—CH₂—, and any analogs of phosphate whereinthe phosphorous atom is in the +5 oxidation state and one or more of theoxygen atoms is replaced with a non-oxygen moiety, e.g., sulfur. Apeptide nucleic acid is a nucleic acid analog in which the backbonecomprises synthetic peptide like linkages (amide bonds) usually formedfrom N-(2-amino-ethyl)-glycine units, resulting in an achiral anduncharged molecule. Exemplary phosphate analogs include associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions are present.

In some embodiments, nucleotides include modified base portionsincluding, but not limited to, pyrimidine nucleobases and purinenucleobases, and derivatives and analogs thereof, including but notlimited to, pyrimidines and purines substituted by one or more of analkyl, caboxyalkyl, amino, hydroxyl, halo (i.e., fluoro, chloro, bromo,or iodo), thiol or alkylthiol moiety. Alkyl (e.g., alkyl, caboxyalkyl,etc.) moieties comprise from 1 to 6 carbon atoms. Further modifiednucleotides contemplated herein include an oxetane-modified base (for adiscussion of base constraining oxetane modifications, see U.S.Published Patent Application No. 20040142946).

Pyrimidines, pyrimidine derivatives and pyrimidine analogs include, butare not limited to, bromothymine, 1-methylpseudouracil, 2-thiouracil,2-thiothymine, 2-thiocytosine, 2-thiopyrimidine,2-hydroxy-5-methyl-4-triazolopyridine, 3-methylcytosine,3-(3-amino-3-carboxy-propyl)uracil, 4-acetylcytosine, 4-thiouracil,N4,N4-ethanocytosine, 4-(6-aminohexylcytosine), 5-methylcytosine,5-ethylcytosine, 5-(C3,C6)-alkynylcytosine, 5-bromouracil,5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil,5-chlorouracil, 5-ethyluracil, 5-fluorouracil, 5-iodouracil,5-propyluracil, 5-propynyluracil, thiouracil,5-carboxymethylaminomethyluracil, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, 5-methoxycarbonylmethyluracil,5-methoxyuracil, uracil-5-oxyacetic acid-methylester, uracil-5-oxyaceticacid, 5-methyl-2-thiouracil, 5-iodo-2′-deoxyuracil, 5-fluorouracil,5-methyluracil, tricyclic carbazole-based pyrimidine analogs, tricyclicphenoxazine-based pyrimidine analogs, isocytosine, pseudoisocytosine,dihydrouracil, pseudouracil and universal nucleotides.

In some embodiments, purines, purine derivatives and purine analogsinclude, but are not limited to, azapurine, azaguanine, azaadenine,deazapurine, deazaguanine, deazaadenine, 1-methylguanine,1-methyladenine, 1-methylinosine, 2-aminopurine, 2-chloro-6-aminopurine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,2-methylthio-N6-isopentenyladenine, 2,6-diaminopurine, 6-aminopurine,6-thioguanine, 6-thioadenine, 6-thiopurine, 6-hydroxyaminopurine,N6-methyladenine, N,N-diemethyladenine, N6-isopentenyladenine,N6,N6-ethano-2,6-diaminopurine, 7-deazaxanthine, 7-deazaguanine,7-methylguanine, 7-halo-7-deaza purine where halo is bromo, fluoro, iodoor chloro, 7-propyne-7-deaza purine, 8-bromoadenine, 8-hydroxyadenine,8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-hydroxyguanine,8-methylguanine, 8-thioguanine, 8-oxo-N6-methyladenine,N-((9-β-D-ribofuranosylpurine-6-yl)-carbamoyl)threonine,methylthioadenine, xanthine, hypoxanthine, inosine, wybutoxosine,wybutosine, isoguanine, queuosine, β-D-mannosylqueuosine,β-D-galactosylqueuosine, and universal nucleotides.

A universal base may base pair with more than one type of specific baseor with any specific base. In other embodiments, a universal basedoesn't hydrogen bond specifically with any base but interacts withadjacent bases on the same nucleic acid strand by hydrophobic stacking.Universal bases include, but are not limited to, indoles such as7-azaindole, 6-methyl-7-azaindole, propynyl-7-aza-indole,allenyl-7-azaindole, isocarbostyril, propynylisocarbostyril,imidizopyridine, and pyrrollpyrizine.

In some embodiments, modified nucleotides are present in siRNAs inparticular patterns, termed herein, “formats,” as exemplified by thepatterns or formats set forth by FIG. 1A-FIG. 1K. In some embodiments, achemically synthesized passenger (sense) oligonucleotide has a length of15 to 30 nucleotides and comprises one of sequence-independentmodification format (1), sequence-independent modification format (2),and sequence-independent modification format (3):

-   -   (1) 5′ N_(p)-m-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein p is 0 or 1;        x is 7, 8, 9, 10, or 11; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus (p+x+r+4);    -   (2) 5′ N_(p)-m-m-N_(y)-m-N_(z)-m-N_(q)-n_(r) 3′, wherein p is 0        or 1; y is 7, 8, or 9 and z is 1; or y is 7 and z is 2 or 3; r        is 0, 1, or 2; and q is an integer representing the length of        the passenger strand minus (p+y+z+r+4);    -   (3) 5′ m-N₁-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein x is 8, 9, or        10; r is 0, 1, or 2; and q is an integer representing the length        of the passenger strand minus (x+r+5);        wherein each m is independently a bicyclonucleotide or a        tricyclonucleotide; each n is independently a deoxynucleotide,        modified nucleotide, or ribonucleotide; and when m is a        bicyclonucleotide, each N is independently a nucleotide other        than a bicyclonucleotide; and when m is a tricyclonucleotide,        each N is independently a nucleotide other than a        tricyclonucleotide.

In some embodiments, a chemically synthesized passenger (sense)oligonucleotide is provided, the oligonucleotide consists essentially ofa length of 15 to 30 nucleotides and consists essentially of one ofsequence-independent modification format (1), sequence-independentmodification format (2), and sequence-independent modification format(3) as follows:

-   -   (1) 5′ N_(p)-m-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein p is 0 or 1;        x is 7, 8, 9, 10, or 11; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus (p+x+r+4);    -   (2) 5′ N_(p)-m-m-N_(y)-m-N_(z)-m-N_(q)-n_(r) 3′, wherein p is 0        or 1; y is 7, 8, or 9 and z is 1; or y is 7 and z is 2 or 3; r        is 0, 1, or 2; and q is an integer representing the length of        the passenger strand minus (p+y+z+r+4);    -   (3) 5′ m-N₁-m-N_(x)-m-m-N_(q)-n_(r) 3′, wherein x is 8, 9, or        10; r is 0, 1, or 2; and q is an integer representing the length        of the passenger strand minus (x+r+5);        wherein each m is independently a bicyclonucleotide or a        tricyclonucleotide; each n is independently a deoxynucleotide,        modified nucleotide, or ribonucleotide; when m is a        bicyclonucleotide, each N is independently a nucleotide other        than a bicyclonucleotide, and when m is a tricyclonucleotide,        each N is independently a nucleotide other than a        tricyclonucleotide.

In some embodiments, a guide (antisense) oligonucleotide comprises 15 to30 nucleotides, a region of continuous complementarity to the passenger(sense) oligonucleotide of at least 12 nucleotides, and hascomplementarity to at least a portion of a target mRNA. In someembodiments, a guide (antisense) oligonucleotide comprises a region ofcontinuous complementarity to the passenger (sense) oligonucleotide offrom at least 13 nucleotides to a number of nucleotides that correspondsto the full length of the oligonucleotide. In some embodiments, a guide(antisense) oligonucleotide comprises a region of continuouscomplementarity to the full length of the passenger (sense)oligonucleotide with the exception of the two 3′ nucleotides.

In some embodiments, the guide (antisense) oligonucleotide hascomplementarity of at least 12 continuous nucleotides to at least aportion of a target mRNA. In some embodiments, the guide (antisense)oligonucleotide has complementarity of at least from 13 nucleotides to anumber of nucleotides that corresponds to the full length of the guideoligonucleotide to at least a portion of a target mRNA. In someembodiments, a guide oligonucleotide has full complementarity to aportion of a target mRNA with the exception of the two 3′ nucleotides.

In some embodiments, a short interfering RNA is provided that comprisesa passenger (sense) oligonucleotide having a length of 17 to 30nucleotides and comprising one of sequence-independent modificationformat (4), format (5), and format (6) and a guide (antisense)oligonucleotide having 17 to 30 nucleotides, a region of continuouscomplementarity to the passenger (sense) oligonucleotide of at least 12nucleotides; the guide strand further having complementarity to at leasta portion of target mRNA:

-   -   (4) 5′ m_(p)-N_(x)-m-m-N_(q)-n_(r) 3′ wherein when p is 0, x is        12; when p is 1, x is 11; when p is 2, x is 10; when p is 3, x        is 9, when p is 4, x is 8; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus (p+x+r+2);    -   (5) 5′ m-m-N_(x)-m-N_(q)-n_(r) 3′ wherein x is 11; r is 0, 1, or        2; and q is an integer representing the length of the passenger        strand minus (x+r+3);    -   (6) 5′ m_(p)-N_(y)-m-N_(z)-m-N_(q)-n_(r) 3′, wherein p is 3; y        is 9 and z is 1; r is 0, 1, or 2; and q is an integer        representing the length of the passenger strand minus        (p+y+z+r+2);        wherein each m is independently a bicyclonucleotide, a        tricyclonucleotide, or a 2′-modified nucleotide; each n is        independently a deoxynucleotide, modified nucleotide, or        ribonucleotide and is an overhanging nucleotide; when m is a        bicyclonucleotide, each N is independently a nucleotide other        than a bicyclonucleotide, when m is a tricyclonucleotide, each N        is independently a nucleotide other than a tricyclonucleotide,        and when m is a 2′-modified nucleotide, each N is independently        a nucleotide other than a 2′-modified nucleotide.

In an embodiment of the short interfering RNA, the passenger (sense)oligonucleotide has sequence independent modification format (4), p is2, x is 10, r is 2, each n is an overhanging nucleotide, and each n isindependently a modified nucleotide; the guide (antisense)oligonucleotide comprises two 3′-overhanging modified nucleotides; andeach modified nucleotide is independently a bicyclonucleotide, atricyclonucleotide, or a 2′-modified nucleotide. In one embodiment, atleast one of the 3′ antepenultimate and the 3′ preantepenultimatepositions of the passenger oligonucleotide is a modified nucleotide. The3′ antepenultimate position is the third from the last position, i.e.,the position prior to the penultimate position, e.g., as for Format DH47of FIG. 1H. The 3′ preantepenultimate position is the fourth from thelast position, i.e., the position prior to the antepenultimate position,e.g., as for Format DH29 of FIG. 1H. Such siRNAs are particularly stableto nucleases such as in the presence of serum, for example.

In some embodiments of the short interfering RNA, when the length is 17nucleotides, at least one of positions 2 and 3 of the guideoligonucleotide is a modified nucleotide; when the length is 18nucleotides, at least one of positions 2, 3 and 4 of the guideoligonucleotide is a modified nucleotide; when the length is 19nucleotides, at least one of positions 2, 3, 4 and 5 of the guideoligonucleotide is a modified nucleotide; when the length is 20nucleotides, at least one of positions 2, 3, 4, 5 and 6 of the guideoligonucleotide is a modified nucleotide; and when the length is 21-30nucleotides, at least one of positions 2, 3, 4, 5, 6 and 7 of the guideoligonucleotide is a modified nucleotide. Formats DH1, and FormatsDH39-DH43 of FIG. 1K provide examples of siRNA modification formatshaving a length of 21 nucleotides and having at least one of positions2, 3, 4, 5, 6 and 7 of the guide oligonucleotide as a modifiednucleotide. Such siRNAs are also particularly stable to nucleases suchas in the presence of serum or plasma, for example.

In some embodiments, the passenger (sense) oligonucleotide comprisessequence-independent modification format (1) where p is 0, x is 10 and ris 2 (Format H, a 21-mer having Format H is depicted in FIG. 1A); orsequence-independent modification format (1) where p is 0, x is 9 or 11,and r is 2 (Format H−1 or Format H+1, 21-mers having such formats aredepicted in FIG. 1B); or sequence-independent modification format (2)where p is 0, y is 9, and r is 2 (Format V, a 21-mer having Format V isdepicted in FIG. 1C).

In some embodiments, the passenger (sense) oligonucleotide comprisessequence-independent modification format (1) where p is 1, x is 9 or 10,r is 2, (Format W or Format W+1, 21-mers having such formats aredepicted in FIG. 1D) and each m has structure (a) where R1 is O, R2 isO, and R3 is CH₂. In some embodiments, the passenger (sense)oligonucleotide comprises sequence-independent modification format (3),x is 9 or 10, r is 2, (Format Y or Format Y+1, 21-mers having suchformats are depicted in FIG. 1D) and each m has structure (a) where R1is O, R2 is O, and R3 is CH₂.

In further embodiments, each N of the guide (antisense) strand is anucleotide other than a bicyclonucleotide. In some embodiments, theguide (antisense) strand has no modifications.

In some embodiments, the siRNA passenger oligonucleotide hasmodification Format H wherein modified nucleotides m are at positions 1,2, 13, and 14 of the format; each m is an LNA®; each n is adeoxynucleotide; and each N is a ribonucleotide. In some embodiments,the siRNA passenger oligonucleotide has the modification Format H andeach m has structure (a) where R1 is O, R2 is O, and R3 is CH₂.

In some embodiments, the siRNA passenger oligonucleotide hasmodification Format V wherein modified nucleotides m are at positions 1,2, 12, and 14 of the format; each m is an LNA®; each n is adeoxynucleotide; and each N is a ribonucleotide. In some embodiments,the siRNA passenger oligonucleotide has the modification Format V andeach m has structure (a) where R1 is O, R2 is O, and R3 is CH₂. In someembodiments, the siRNA passenger oligonucleotide has modification FormatV−2 having format (2) where p is 0 and y is 7.

In some embodiments, the siRNA passenger oligonucleotide hasmodification Format Q wherein modified nucleotides m are at positions 13and 14 of the format; each m is an LNA®; each n is a deoxynucleotide;and each N is a ribonucleotide. In some embodiments, the siRNA passengeroligonucleotide has the modification Format Q and each m has structure(a) where R1 is O, R2 is O, and R3 is CH₂.

In some embodiments, the siRNA passenger oligonucleotide has format (4)wherein when p is 0, x is 12 (Format Q); when p is 1, x is 11 (FormatJB2); when p is 2, x is 10 (Format H); when p is 3, x is 9 (Format JB5),and when p is 4, x is 8 (Format JB3) and r is 2. In some embodiments,the siRNA passenger oligonucleotide has format (5) wherein x is 11(Format JB1) and r is 2. In further embodiments, the siRNA passengeroligonucleotide has format (6) wherein p is 3, y is 9, and z is 1(Format JB4) and r is 2.

In some embodiments, the chemically synthesized short interfering RNAcomprises a passenger (sense) oligonucleotide and a guide (antisense)oligonucleotide covalently bonded via nucleotide loop or a linker loopwhich forms a short hairpin oligonucleotide.

In some embodiments, each 3′ end of the siRNA independently has a one,two, or three nucleotide overhang and in some embodiments, each 3′ endhas a two nucleotide overhang wherein the nucleotides of each overhangcomprise deoxyribonucleotides. In some embodiments, at least oneoverhanging nucleotide is a modified nucleotide. In some embodiments,each 3′ end of the siRNA has a two nucleotide overhang and one, two,three or all four of the overhanging nucleotides are modifiednucleotides. In some embodiments, at least one internucleoside linkageis other than a phosphodiester internucleoside linkage.

Chemical Synthesis: Interfering RNA oligonucleotide synthesis isperformed according to standard methods. Non-limiting examples ofsynthesis methods include in vitro chemical synthesis using a diestermethod, a triester method, a polynucleotide phosphorylase method and bysolid-phase chemistry. These methods are discussed in further detailbelow.

Oligonucleotides having bicyclonucleotide structures including those ofstructure (a) are synthesized according to Imanishi as described in U.S.Pat. Nos. 6,770,748 and 6,268,490; according to Wengel as described inU.S. Pat. Nos. 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499,and 6,670,461 and U.S. Published Application No's 2005/0287566, and2003/0224377; according to Kochkine as described in U.S. Pat. Nos.6,734,291, and 6,639,059 and U.S. Published Application No 2003/0092905;and according to Kauppinen as described in U.S. Published ApplicationNo's 2004/0219565, and according to Srivastava, P., et al. (J. Am. Chem.Soc. 129, 8362-8379, 2007) for example.

Oligonucleotides having tricyclonucleotide structure (b) are synthesizedaccording to Ittig, D. et al. (Nucleic Acids Res. 32:1, 346-353, 2004),for example. Oligonucleotides having bicyclonucleotide structure (c) aresynthesized according to Maier, M. A., et al. (Nucleic Acids Res. 32:12,3642-3650, 2004), for example. Oligonucleotides having a FANAsubstituent are synthesized according to Dowler et al. (Nucleic AcidsRes. 2006, 34:6, p 1669-1675).

The diester method of oligonucleotide synthesis was the first to bedeveloped to a usable state, primarily by Khorana et al. (Science, 203,614. 1979). The basic step is the joining of two suitably protectednucleotides to form a dinucleotide containing a phosphodiester bond. Thediester method is well established and has been used to synthesize DNAmolecules.

The main difference between the diester and triester methods is thepresence in the latter of an extra protecting group on the phosphateatoms of the reactants and products (Itakura et al., J. Biol. Chem.,250:4592 1975). The phosphate protecting group is usually a chlorophenylgroup, which renders the nucleotides and polynucleotide intermediatessoluble in organic solvents. Therefore purifications are done inchloroform solutions. Other improvements in the method include (i) theblock coupling of trimers and larger oligomers, (ii) the extensive useof high-performance liquid chromatography for the purification of bothintermediate and final products, and (iii) solid-phase synthesis.

Drawing on the technology developed for the solid-phase synthesis ofpolypeptides, it has been possible to attach the initial nucleotide tosolid support material and proceed with the stepwise addition ofnucleotides. All mixing and washing steps are simplified, and theprocedure becomes amenable to automation. These syntheses are nowroutinely carried out using automatic nucleic acid synthesizers.

Phosphoramidite chemistry (Beaucage, S. L. and Iyer, R. P. Tetrahedron,1993(49) 6123; Tetrahedron, 1992(48) 2223) has become by far the mostwidely used coupling chemistry for the synthesis of oligonucleotides. Asis well known to those skilled in the art, phosphoramidite synthesis ofoligonucleotides involves activation of nucleoside phosphoramiditemonomer precursors by reaction with an activating agent to formactivated intermediates, followed by sequential addition of theactivated intermediates to the growing oligonucleotide chain (generallyanchored at one end to a suitable solid support) to form theoligonucleotide product.

A siRNA may be purified on reversed phase purification cartridges, ionexchange HPLC, agarose gels, polyacrylamide gels, cesium chloridecentrifugation gradients, a column, filter, or cartridge containing anagent that binds to the nucleic acid, such as a glass fiber, or by anyother means known to one of ordinary skill in the art. Gelelectrophoresis can be used for determining single vs. double strandstructure, ion exchange HPLC can be used for determining purity,MALDI-MS can be used for determining identity, and UV spectroscopy canbe used for quantitative determinations of siRNAs.

A cell containing an endogenous target gene may be derived from orcontained in any organism (e.g., eukaryotes such as plants, animals,fungi; prokaryotes such as bacteria). The plant may be a monocot, dicotor gynmosperm; the animal may be a vertebrate or invertebrate. A microbemay be used in agriculture or by industry, or may be pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies. Examples of vertebrates include fish and mammals,including cattle, goat, pig, sheep, hamster, mouse, rats and humans;invertebrate animals include nematodes, insects, arachnids, and otherarthropods.

The target gene can be a gene derived from a cell, an endogenous gene, atransgene, or exogenous genes such as genes of a pathogen, for example,a virus, which is present in the cell after infection thereof. The cellhaving the target gene may be from the germ line or somatic, totipotentor pluripotent, dividing or non-dividing, parenchyma or epithelium,immortalized or transformed, or the like. The cell can be a gamete or anembryo; if an embryo, it can be a single cell embryo or a constituentcell or cells from a multicellular embryo. The term “embryo” thusencompasses fetal tissue. The cell having the target gene may be anundifferentiated cell, such as a stem cell, or a differentiated cell,such as from a cell of an organ or tissue, including fetal tissue, orany other cell present in an organism. Cell types that aredifferentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine orexocrine glands.

In some embodiments, the target RNA is transcribed RNA, including codingRNA and noncoding RNA.

As used herein, the terms “cell,” “cell line,” and “cell culture”include their progeny, which is any and all subsequent generationsformed by cell division. It is understood that all progeny may not beidentical due to deliberate or inadvertent mutations. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a small, interfering RNA or atemplate construct encoding such an RNA has been introduced. Therefore,recombinant cells are distinguishable from naturally occurring cellswhich do not contain a recombinantly introduced nucleic acid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

In some embodiments, a tissue containing an endogenous target gene ishuman tissue. In certain embodiments, the tissue comprises blood, forexample but not limited to, red blood cells, white blood cells,platelets, plasma, serum, or whole blood. In certain embodiments, thetissue comprises a solid tissue. In certain embodiments, the tissuecomprises a virus, bacteria, or fungus. In certain embodiments, thetissue comprises ex vivo tissue.

A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to, blood (e.g., hematopoietic cells (such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34+cells CD4+cells), lymphocytes and other blood lineage cells), bonemarrow, brain, stem cells, blood vessel, liver, lung, bone, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, skin, small intestine,spleen, stomach, testes.

In some embodiments, modification-formatted siRNAs are provided that arestabilized to intracellular and extracellular nucleases. Such siRNAs aredemonstrated herein to remain at full-length in the presence ofbiological fluids for a longer time than previously known stabilizedsiRNAs. Nuclease stabilized siRNAs are useful in biological fluids andtissues that contain particularly high concentrations of nucleasesincluding, for example, blood serum, blood plasma, and organs andtissues that are particularly vascularized.

A siRNA may be associated with a cell-targeting ligand. As used herein,a “cell targeting ligand” is a cell-directing molecule that hasspecificity for targeted sites such as cell surface receptors.“Specificity for targeted sites” means that upon contacting the celltargeting ligand with a cell, under physiological conditions of ionicstrength, temperature, pH and the like, specific binding will occur. Thecell-ligand interaction may occur due to specific electrostatic,hydrophobic, or other interaction of certain residues of the ligand withspecific residues of the cell to form a stable complex under conditionseffective to promote the interaction.

Exemplary cell targeting ligands include, but are not limited to,polynucleotides, oligonucleotides, polyamides, peptides having affinityfor a cellular receptor, proteins such as antibodies, fatty acids,vitamins, flavonoids, sugars, antigens, receptors, reporter molecules,reporter enzymes, chelators, porphyrins, intercalators, steroids andsteroid derivatives, hormones such as progestins (e.g. progesterone),glucocorticoids (e.g., cortisol), mineralocorticoids (e.g.,aldosterone), androgens (e.g., testosterone) and estrogens (e.g.,estradiol), histamine, hormone mimics such as morphine, and macrocycles.A peptide having affinity for a cellular receptor may include anendorphin, an enkephalin, a growth factor, e.g. epidermal growth factor,poly-L-lysine, a hormone, insulin, ribonuclease, serum albumin bindingpeptide, a peptide region of a protein and other molecules that arecapable of penetrating cellular membranes, either by active transport orpassive transport.

Suitable methods for siRNA delivery to effect RNAi according toembodiments include any method by which a siRNA can be introduced intoan organelle, a cell, a tissue or an organism, as described herein or aswould be known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, direct delivery of siRNA such as byinjection including microinjection, electroporation, calcium phosphateprecipitation, using DEAE-dextran followed by polyethylene glycol,direct sonic loading, liposome mediated transfection, microprojectilebombardment, agitation with silicon carbide fibers,Agrobacterium-mediated transformation, PEG-mediated transformation,desiccation/inhibition-mediated uptake, and the like. Through the use oftechniques such as these, an organelle, cell, tissue or organism may bestably or transiently transformed. SiRNAs that are stabilized tonuclease digestion, embodiments of which are provided herein, areparticular suited for direct injection as demonstrated by the data ofExample 8 where serum-stabilized modification-formatted siRNAs werepresent in vivo at a level 400% greater than unmodified siRNAs.

Techniques for visualizing or detecting siRNA include, withoutlimitation, microscopy, arrays, fluorometry, light cyclers or other realtime PCR machines, FACS analysis, scintillation counters,phosphoimagers, Geiger counters, MRI, CAT, antibody-based detectionmethods (Westerns, immunofluorescence, immunohistochemistry),histochemical techniques, HPLC, spectroscopy, mass spectroscopy;radiological techniques, capillary gel electrophoresis, and mass balancetechniques. Alternatively, nucleic acids may be labeled or tagged toallow for their efficient isolation. In other embodiments of theinvention, nucleic acids are biotinylated.

A “label” or “reporter,” refers to a moiety or property that allows thedetection of that with which it is associated. The label can be attachedcovalently or non-covalently. Examples of labels include fluorescentlabels (including, e.g., quenchers or absorbers), colorimetric labels,chemiluminescent labels, bioluminescent labels, radioactive labels,mass-modifying groups, antibodies, antigens, biotin, haptens, enzymes(including, e.g., peroxidase, phosphatase, etc.), and the like.Fluorescent labels can include dyes that are negatively charged, such asdyes of the fluorescein family including, e.g. FAM, HEX, TET, JOE, NANand ZOE; or dyes that are neutral in charge, such as dyes of therhodamine family including, e.g., Texas Red, ROX, R110, R6G, and TAMRA;or dyes that are positively charged, such as dyes of the cyanine familyincluding e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, and TAMRA are available from, e.g.,Perkin-Elmer, Inc. (Wellesley, Mass.); Texas Red is available from,e.g., Molecular Probes, Inc. (Eugene, Oreg.); and Cy2, Cy3, Cy3.5, Cy5,Cy5.5 and Cy7, and are available from, e.g., Amersham Biosciences Corp.(Piscataway, N.J.). In certain embodiments, the fluorescer molecule is afluorescein dye and the quencher molecule is a rhodamine dye.

A label or reporter can comprise both a fluorophore and a fluorescencequencher. The fluorescence quencher can be a fluorescent fluorescencequencher, such as the fluorophore TAMRA, or a non-fluorescentfluorescence quencher (NFQ), for example, a combined NFQ-minor groovebinder (MGB) such as an MGB ECLIPSE™ minor groove binder supplied byEpoch Biosciences (Bothell, Wash.) and used with TAQMAN™ probes (AppliedBiosystems, Foster City, Calif.). The fluorophore can be any fluorophorethat can be attached to a nucleic acid, such as, for example, FAM, HEX,TET, JOE, NAN, ZOE, Texas Red, ROX, R110, R6G, TAMRA, Cy2, Cy3.5, Cy5,Cy5.5 and Cy7 as cited above as well as VIC, NED, LIZ, ALEXA, Cy9, anddR6G.

Further examples of labels include black hole quenchers (BHQ)(Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), andDabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch). Labels canalso comprise sulfonate derivatives of fluorescein dyes, phosphoramiditeforms of fluorescein, phosphoramidite forms of CY5 (available forexample from Amersham), intercalating labels such as ethidium bromide,and SYBR™ Green I and PICOGREEN™ (Molecular Probes).

In various embodiments, detection of fluorescence can be by any methodknown to skilled artisans, and can be qualitative or quantitative.Quantitative results can be obtained, for example, with the aid of afluorimeter. In some embodiments, detection can be achieved usingmicroarrays and related software such as the Applied Biosystems ArraySystem with the Applied Biosystems 1700 Chemiluminescent MicroarrayAnalyzer and other commercially available array systems available fromAffymetrix, Agilent, Illumina, and NimbleGen, among others (see alsoGerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., MinervaBiotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, includingsupplements, 2003).

A biologically acceptable carrier refers to those carriers that providesuitable preservation, if needed, and deliver one or more interferingRNAs of the present embodiments in a homogenous dosage. The type ofcarrier may depend on whether the siRNA is to be used in a solid, liquidor aerosol form, and whether it needs to be sterile for such routes ofadministration as injection. The siRNA compositions may be formulatedinto a composition in a free base, neutral or salt form. A salt form ofsiRNA has properties of reducing off-target effects and maintainingknockdown activity comparable to that of the siRNAs described herein.Examples of salt forms include organic acid addition salts and baseaddition salts. SiRNA compositions may be provided in a prodrug formthat can be metabolized intracellularly into the active form; suchprodrug forms may include a protection group such as anS-acetylthioethyl or S-pivaloylthioethyl group.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to,RNase-free water, buffer, saline, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g.,triglycerides, vegetable oils, liposomes), glycine, acceptablepreservatives (e.g., antibacterial agents, antifungal agents),co-solvents, surfactants, penetration enhancers (e.g., cremephor andTWEEN™ 80 (polyoxyethylene sorbitan monolaureate, Sigma Aldrich, St.Louis, Mo.)), hyaluronic acid, mannitol, benzalkonium chloride,viscosity building agents (e.g., hydroxymethyl cellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone,hydroxypropylmethylcellulose), coatings, surfactants, antioxidants,isotonic agents, absorption delaying agents, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and combinations thereof. Sterile injectable solutions areprepared by incorporating the siRNA in an appropriate solvent withvarious of the other ingredients enumerated above, as desired, followedby filtered sterilization. Formulations comprise interfering RNAs, orsalts thereof, of embodiments herein up to 99% by weight mixed with abiologically acceptable carrier. Interfering RNAs of embodiments can beemployed as solutions, suspensions, or emulsions.

An acceptable carrier for use of interfering RNA of embodiments includesthe lipid-based reagent siPORT™ NeoFX™ Transfection Agent (Ambion Cat.#AM4510), the polyamine-based reagent siPORT™ Amine Transfection Agent(Ambion Cat. #AM4502), the cationic and neutral lipid-based reagentsiPORT™ Lipid Transfection Agent (Ambion Cat. #AM4504), the cationiclipid-based transfection reagent TransIT®-TKO (Mirus Corporation,Madison, Wis.), Lipofectin®, Lipofectamine, Oligofectamine™ (Invitrogen,Carlsbad, Calif.), or Dharmafect™ (Dharmacon, Lafayette, Colo.), furtherpolycations such as polyethyleneimine, cationic peptides such as Tat,polyarginine, or penetratin, or liposomes. Liposomes are formed fromstandard vesicle-forming lipids and a sterol, such as cholesterol, andmay include a targeting molecule such as a monoclonal antibody havingbinding affinity for a cell surface antigen, for example. Further, theliposomes may be PEGylated liposomes.

Interfering RNAs may be delivered in solution, in suspension, or inbioerodible or non-bioerodible delivery devices. The interfering RNAscan be delivered alone or as components of defined, covalent conjugatessuch as with a polyethylene glycol moiety, a cholesterol moiety, or withgrowth factors for receptor-mediated endocytosis. The interfering RNAscan also be complexed with cationic lipids, cationic peptides, orcationic polymers; complexed with proteins, fusion proteins, or proteindomains with nucleic acid binding properties (e.g., protamine); orencapsulated in nanoparticles or liposomes. Tissue- or cell-specificdelivery can be accomplished by the inclusion of an appropriatetargeting moiety such as an antibody or antibody fragment. Exceptinsofar as any conventional carrier is incompatible with the siRNA, itsuse in a composition, or as a biological carrier is contemplated byembodiments herein.

Interfering RNA of embodiments herein can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraventricularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrauterinely, intrarectally, topically,intratumorally, intrathecally, intramuscularly, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g. aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the foregoing aswould be known to one of ordinary skill in the art (Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co. 1990).

A dosage for ex vivo or in vivo use is from 0.01 μg to 1 g/kg of bodyweight, and may be administered once or more per day, week, month oryear. Generally, an effective amount of the interfering RNAs havingmodified nucleotides and modification formats of embodiments herein isan amount sufficient to reduce off-target events while maintainingpotency and results in an extracellular concentration at the surface ofthe target cell of from 100 pM to 1 μM, or from 1 nM to 100 nM, or from2 nM to about 25 nM, or to about 10 nM. The amount required to achievethis local concentration will vary depending on a number of factorsincluding the delivery method, the site of delivery, the number of celllayers between the delivery site and the target cell or tissue, whetherdelivery is local or systemic, etc. The concentration at the deliverysite may be considerably higher than it is at the surface of the targetcell or tissue. The pH of the formulation is about pH 4-9, or pH 4.5 topH 7.4.

In some embodiments, the ability of siRNA to knockdown the levels ofendogenous target gene expression is evaluated in vitro as follows.Cells are transfected using the siPORT™ NeoFX™ Transfection Agent(Ambion/Applied Biosystems Austin, Tex. Cat #AM4510) using the “reverse”transfection protocol described by the manufacturer. Unmodified controlor modification-formatted siRNA is plated into 96-well tissue cultureplates to achieve a final concentration of, for example, either 1 nM or5 nM. SiPORT™ NeoFX™ transfection agent is complexed with siRNA for atleast 10 minutes, followed by overlaying freshly trypsinized cells(about 4.0×10³) on the siRNA/transfection agent complex. The plates areincubated under tissue culture conditions at 37° C. for 48 hours.

After harvesting transfected cells, total RNA is isolated using theMagMAX™-96 total RNA isolation kit (Ambion, Inc. Austin Tex. Cat.#AM1830) according to the manufacturer's protocol. RNA is eluted in 50μl of nuclease-free water. CDNA is generated using the High CapacitycDNA Reverse Transcription Kit (Applied Biosystems, Inc., Foster CityCalif., Cat. #4368814) according to the manufacturer's protocol in a 30μl reaction volume. TaqMan® Gene Expression Assays (Applied Biosystems,Inc., Cat. #4331182) are carried out using 2 μl of cDNA in a 10 μl totalPCR reaction volume. Technical replicates are generated and an 18S valueis calculated for each sample. Preamplification uniformity is checked bycalculating the ddCT using the following formula:ddCT=(siRNA treated Ct_(GOI)−siRNA treated Ct_(18S))−(Neg controlCt_(GOI)−Neg control Ct_(18S))GOI=endogenous gene of interest18S=18S ribosomal RNA normalizersiRNA treated=biological sample treated with an siRNANeg control=biological sample treated with an siRNA that has no homologyto any known target mRNA

The % remaining was calculated using the following formula:% remaining=100×2^(−(ddCT)).

Target protein levels may be assessed approximately 72 hpost-transfection (actual time dependent on protein turnover rate) bywestern blot, for example. Standard techniques for RNA and/or proteinisolation from cultured cells are well-known to those skilled in theart.

Techniques for visualizing or detecting siRNA include those cited above.Nucleic acids may be labeled or tagged to allow for their efficientisolation. Thus, the silencing ability of any given siRNA can be studiedby one of any number of art tested procedures.

A “kit,” as used herein, refers to a combination of at least some itemsfor reducing off-target effects in RNA interference. Embodiments of kitscomprise, for example, at least one siRNA having modified nucleotides ina modification format of embodiments herein. The at least one siRNA canbe custom made. In some embodiments, kits comprise at least one siRNAhaving bicyclonucleotides in modification Format H, Q, V−2, Y, Y+1, JB1,JB2, JB3, JB4 or JB5 together with a biologically acceptable carriersuch as a buffer, and a transfection agent or other suitable deliveryvehicle.

Embodiments of kits can further comprise a validated positive controlsiRNA that targets a housekeeping gene in all cell types of human,mouse, and rat. Embodiments of kits can further comprise a validatednegative control siRNA that is nontargeting. The siRNA controls may bepre-plated and may be labeled with a detectable marker.

Embodiments of kits can further comprise reagents for assessingknockdown of the intended target gene such as antibodies for monitoringknockdown at the protein level by immunofluorescence or Westernanalysis, reagents for assessing enzymatic activity or presence of areporter protein, or reagents for assessing cell viability. RT-PCRprimers and probes may be included for detection of target or reportermRNA. Embodiments of kits can further comprise an RNase inhibitor.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other packaging means, intowhich a component can be placed, and in some embodiments, suitablyaliquoted. Where more than one component is included in the kit (theycan be packaged together), the kit also will generally contain at leastone second, third or other additional container into which theadditional components can be separately placed. However, variouscombinations of components can be packaged in a container means. Thekits of the present teachings also will typically include a means forcontaining the siRNA having modified nucleotides in a modificationformat as set forth herein, and any other reagent containers in closeconfinement for commercial sale. Such containers can include injectionor blow-molded plastic containers into which the desired container meansare retained. When the components of the kit are provided in one and/ormore liquid solutions, the liquid solution comprises an aqueous solutionthat can be a sterile aqueous solution.

In certain embodiments, at least one kit component is lyophilized andprovided as dried powder(s). When reagents and/or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. In certain embodiments, the solvent isprovided in another container means. Kits can also comprise anadditional container means for containing a sterile, biologicallyacceptable buffer and/or other diluent.

A kit can also include instructions for employing the kit components aswell as the use of any other reagent not included in the kit.Instructions can include variations that can be implemented.

The present method and kit embodiments may be employed for high volumescreening. A library of either siRNA or candidate siRNA can beconstructed using embodiments herein. This library may then be used inhigh throughput assays, including microarrays. Specifically contemplatedare chip-based nucleic acid technologies that involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Byusing fixed probe arrays, one can employ chip technology to segregatetarget molecules as high density arrays and screen these molecules onthe basis of hybridization. The term “array” as used herein refers to asystematic arrangement of nucleic acid. For example, a nucleic acidpopulation that is representative of a desired source (e.g., human adultbrain) is divided up into the minimum number of pools in which a desiredscreening procedure can be utilized to detect or deplete a target geneand which can be distributed into a single multi-well plate. Arrays maybe of an aqueous suspension of a nucleic acid population obtainable froma desired mRNA source, comprising: a multi-well plate containing aplurality of individual wells, each individual well containing anaqueous suspension of a different content of a nucleic acid population.Examples of arrays, their uses, and implementation of them can be foundin U.S. Pat. Nos. 6,329,209, 6,329,140, 6,324,479, 6,322,971, 6,316,193,6,309,823, 5,412,087, 5,445,934, and 5,744,305, which are hereinincorporated by reference.

Microarrays are known in the art and consist of a surface to whichprobes that correspond in sequence to gene products (e.g., cDNAs, mRNAs,cRNAs, polypeptides, and fragments thereof), can be specificallyhybridized or bound at a known position. In one embodiment, themicroarray is an array (i.e., a matrix) in which each positionrepresents a discrete binding site for a product encoded by a gene(e.g., a protein or RNA), and in which binding sites are present forproducts of most or almost all of the genes in the organism's genome. Ina preferred embodiment, the “binding site” is a nucleic acid or nucleicacid analogue to which a particular cognate cDNA can specificallyhybridize. The nucleic acid or analogue of the binding site can be,e.g., a synthetic oligomer, a full-length cDNA, a less-than full lengthcDNA, or a gene fragment.

A solid support may be made from glass, plastic (e.g., polypropylene,nylon), polyacrylamide, nitrocellulose, or other materials. Exemplarymethods for attaching nucleic acids to a surface include printing onglass plates or by masking. In principal, any type of array, forexample, dot blots on a nylon hybridization membrane, could be used,although, as will be recognized by those of skill in the art, very smallarrays will be preferred because hybridization volumes will be smaller.

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1 siRNA Modification Formats

Short interfering RNAs (siRNAs) were chemically synthesized usingstandard phosphoramidite-based nucleoside monomers and established solidphase oligomerization cycles according to Beaucage, S. L. and Iyer, R.P. (Tetrahedron, 1993 (49) 6123; Tetrahedron, 1992 (48) 2223). Synthesisof oligonucleotides was performed on a BioAutomation MerMade 192synthesizer (BioAutomation Corp, Plano, Tex.). Eight equivalents ofactivator were used for every equivalent of LNA® phosphoramidite toprovide a satisfactory stepwise coupling yield of >98% per baseaddition. Purification of the individual siRNA strands was carried outusing either disposable reversed phase purification cartridges or ionexchange HPLC. Gel electrophoresis was used for determining single vs.double strand structure for annealed samples, ion exchange HPLC was usedfor determining single strand purity, MALDI mass spectrometry was usedfor determining single strand identity, and UV spectroscopy was used forquantitative determination for both single strand oligonucleotides andsiRNAs. The guide and passenger strands of each siRNA are both 21nucleotides long and every siRNA was designed with a two base overhangon both 3′ ends. Position 1 of each guide (antisense) strand wasdesigned to be either an “A nucleotide” or a “U nucleotide.” When acytidine (C) nucleoside was modified as a bicyclo-sugar, the substitutednucleobase was a 5-methylated cytidine residue. When a uridine (U)nucleoside was modified as a bicyclo-sugar, the substituted nucleobasewas a thymine (T) residue. The overhanging nucleotides are as depictedin FIG. 1A-FIG. 1K and are (independently): deoxynucleotides, modifiednucleotides, or ribonucleotides. The guide strand was chemicallyphosphorylated in the case of Format “O.” All other 3′-ends and 5′-endswere synthesized to contain hydroxyl groups.

Modified nucleotides introduced into the siRNA molecule included lockednucleic acid (LNA® residues), in particular those having structure (a)above where R1 is O, R2 is O, and R3 is CH₂; 2′-O-methyl nucleotidederivatives; and 2′-5′ linked nucleotides. LNA® amidites were purchasedfrom Proligo (Boulder, Colo.) and Exiqon A/S (Vedbaek, Denmark), and2′-O-methyl nucleotide derivatives and 3′- and 2′-TBDMS RNA amiditeswere purchased from Chemgenes Corporation (Wilmington, Mass.).

Modification formats introduced into siRNAs included those depicted inFIG. 1A (Formats E, F, G, H, J, K, and O in addition to an unmodifiedcontrol, Format A), FIG. 1B (Formats M, H (repeated), H(−1), H(−2),H(+1), and Q), FIG. 1C (Formats M (repeated), H (repeated) V, V(−1),V(−2), V(+1), FIG. 1D (Formats W, W+1, W−1, Y, Y−1, and Y+1), FIG. 1E(Formats JB1, JB2, JB3, JB4, and JB5), and those depicted in FIG.1F-FIG. 1K further described in Example 7.

The modification formats included modifications on only the passenger(sense) strand (Formats E, H, K, M, H−1, H−2, H+1, Q, V, V−1, V−2, V+1,W, W+1, W−1, Y, Y−1, Y+1 JB1, JB2, JB3, JB4, JB5, DH3, DH30, and DH28(See Example 7 for DH formats)) or on both the guide (antisense) strandand the passenger (sense) strand (Formats F, G, J, O, and all DH formatsother than DH3, DH30 and DH28 (See Example 7 for DH formats)). Themodification formats of FIG. 1A-FIG. 1E are further described below.

Modified Positions (Numbered from the 5′ end of each Strand) FormatPassenger Sequence Guide Sequence A, Control None None E 1, 20, 21 NoneF 1, 20, 21 20, 21 G 1, 2, 13, 14 9, 10, 19, 20 H 1, 2, 13, 14 None J 1,2, 10, 14 11, 12, 19, 20 K 1, 2, 10, 14 None O 1, 2 2 plus a 5′phosphateM 1, 2 None H(−1) 1, 2, 12, 13 None H(−2) 1, 2, 11, 12 None H(+1) 1, 2,14, 15 None Q 13, 14 None V 1, 2, 12, 14 None V(−1) 1, 2, 11, 13 NoneV(−2) 1, 2, 10, 12 None V(+1) 1, 2, 13, 15 None W 2, 3, 13, 14 None W +1 2, 3, 14, 15 None W − 1 2, 3, 12, 13 None Y 1, 3, 13, 14 None Y − 1 1,3, 12, 13 None Y + 1 1, 3, 14, 15 None JB1 1, 2, 14 None JB2 1, 13, 14None JB3 1, 2, 3, 4, 13, 14 None JB4 1, 2, 3, 13, 15 None JB5 1, 2, 3,13, 14 None

Format F is previously reported by Mook et al. (Molecular CancerTherapeutics March, 2007 6:3, 833-843). Formats E and F are previouslyreported by Elmen et al. (Nucleic Acids Research Jan. 14, 2005. 33:1,439-447). Format O is described by Jackson et al. (RNA May 8, 2006 12:71197-1205).

Example 2 Assay Methods for siRNA Silencing Effects

Cleavage activity mediated by the guide (antisense) strand and thepassenger (sense) strand of a given siRNA was assayed forexogenously-provided genes as follows. cDNA fragments from genes ofinterest (GOI) were cloned into the pMIR-REPORT™ miRNA ExpressionReporter Vector System (Ambion/Applied Biosystems, Austin Tex. Cat#AM5795) either in the forward or the reverse orientation with respectto the direction of the coding strand of the DNA for the gene using DNAligase and standard cloning techniques. The full length cDNA clones werepurchased from Open Biosystems (Huntsville, Ala.) for the following GOI.

Gene ID Symbol Description 983 CDC2 Cell division cycle 2 1017 CDK2Cyclin-dependent kinase 2 701 BUB1B BUB1 budding uninhibited bybenzimidazoles 1 homolog beta 2222 FDFT1 Farnesyl-diphosphatefarnesyltransferase 1 3156 HMGCR 3-Hydroxy-2methylglutaryl-coenzyme Areductase 7456 WEE1 WEE1 homolog (S. pombe) 5347 PLK1 Polo-like kinase 11213 CLTC Clathrin, heavy chain (Hc) 836 CASP3 Caspase 3,apoptosis-related cysteine peptidase 595 CCND1 Cyclin D1 1595 CYP51A1Cytochrome P450, family 51, subfamily A, polypeptide 1

The coding sequences of the GOI ranged in size from 1.2 kb to greaterthan 6 kb in length. The cDNA fragment from each GOI was subclonedindividually into the HindIII (nucleotide position 463)/SpeI (nucleotideposition 525) multicloning site of the pMIR-REPORT™ miRNA ExpressionReporter Vector that contains firefly luciferase reporter protein underthe control of a mammalian promoter/terminator system as shown in FIG.2A. Standard PCR-based techniques were used to engineer the HindIII andSpeI restriction endonuclease sites. Reporter vectors were generatedthat express a mRNA transcript that included the luciferase mRNA fusedto the mRNA for the GOI in either the forward or the reverse orientationrelative to the fLuc mRNA as shown in FIG. 3A and FIG. 3B. The fLuc-GOIfusion mRNA is expressed under the control of the CMV promoter locatedclosest to the 5′ start of the fLuc coding sequences. When the reporterconstruct is co-transfected into cells with a siRNA complementary to thefLuc or to the cDNA GOI, knockdown as a result of RNA interference ismonitored by either measuring the remaining fLuciferase protein or bythe remaining fLuc-GOI fusion mRNA. The 5′ and 3′ terminal 500nucleotides of all subcloned fragments from each orientation of each GOIwere subjected to nucleotide sequence analysis to verify the identityand orientation of the cDNA contained within the vector. Thus, thesevectors served as the source of substrate for monitoring siRNA cleavagemediated by either the guide strand (forward clone) or the passengerstrand (reverse clone).

The pMIR-REPORT™ Beta-galactosidase Reporter Control Vector(Ambion/Applied Biosystems, Austin Tex. Cat ##AM5795) depicted in FIG.2B was used according to the manufacturer's protocol for normalizationof transfection efficiency. The β-gal coding sequence is expressed underthe control of the CMV promoter. The signal obtained from the fLucreporter is divided by the signal obtained from the respective β-galreporter gene to calculate the normalized luciferase activity for eachassay.

Transfection:

Hela cells (ATCC No. CCL-2, Manassas, Va.) were plated in 96-well tissueculture plates at a density of about 8.0×10³ cells per well. Twenty fourhours later, the cells were transfected using standard transfectionprotocols with siPORT™ Amine Transfection Agent (Ambion/AppliedBiosystems Austin, Tex. Cat #AM4502) along with a final concentration of30 nM of the siRNA under investigation, 40 ng of the pMIR-REPORT™luciferase-GOI plasmid and 4 ng of the pMIR-REPORT™ transfection controlplasmid. Cells were washed with complete media 24 hours posttransfection and were collected for analysis 48 hours aftertransfection. Three biological replicates were tested for each siRNAunder investigation for each plasmid. In parallel, Silencer® Negativecontrol siRNA (Ambion/Applied Biosystems, Austin Tex. Cat #AM4636) wastransfected under the same experimental conditions to measure the basallevel of expression with a non-targeting siRNA for the purposes ofcalculating the % of mRNA knockdown by the passenger and guide strandsfrom test siRNA.

Dual-Light® Combined Reporter Gene Assay System:

The Applied Biosystems Dual-Light® combined reporter gene assay system(Applied Biosystems, Foster City Calif. Cat #T1003) was used tosimultaneously detect luciferase and β-gal in the same sample followingthe manufacturer's recommended protocol. Luminescent readings were takenusing a BMG LABTECH POLARstar Optima reader (BMG LABTECH, Durham, N.C.).

Luciferase assays were performed 2-5 seconds following the addition ofthe luciferin substrate in the Dual-Light® assay system. The plates wereallowed to sit for at least 45 minutes before adding substrates fordetection and measurement of β-galactosidase. Each well was normalizedusing the appropriate β-gal reading to obtain a ratio of luciferasesignal/β-gal signal. The knockdown or % remaining luciferase expressionwas then calculated by normalizing the biological samples against thenegative control siRNA transfected cells.

Example 3 Silencing Effects of siRNA Modification Formats can beStrand-Switched

For this study, two siRNAs for each of three exogenously-provided targetmRNAs were synthesized in the unmodified Format A and for each of 8different test modification Formats (E, F, G, H, J, K, M and O).Therefore, each test format provides data for 6 siRNAs. For the data ofFIG. 4A-FIG. 4E, the modified nucleotides “m” at positions as indicatedby the format letter and referring to FIG. 1A and FIG. 1B were LNA®residues having structure (a) above where R1 is O, R2 is O, and R3 isCH₂. SiRNA sequences chosen for this study illustrated by FIG. 4A wereexperimentally determined to have strand silencing activity on the partof both strands. Such a collection allowed an analysis of the impactthat a modification has for introducing strand bias, i.e., an ability toimpair the silencing activity of one strand while maintaining theactivity of the other strand. For this collection of siRNAs, on average,an about 70% knockdown of the luciferase-GOI mRNA mediated by theunmodified guide strand was demonstrated and, on average, an about 58%knockdown of the luciferase-GOI mRNA mediated by the unmodifiedpassenger strand was demonstrated (FIG. 4A, Format A).

The amount of normalized reporter protein present after carrying outRNAi studies for the unmodified Format A and for each test modificationFormat (E, F, G, H, J, K, M and O) is plotted in FIG. 4A. The dark barsrepresent the % remaining activity due to silencing by the guide strandsince the data result from the clone having the GOI in the forwardorientation. The light bars of FIG. 4A represent the % remainingactivity due to silencing of the passenger strand since the data resultfrom the clone having the GOI in the reverse orientation. Normalizationis carried out by transfection in parallel of a scrambled (nonsense)siRNA, the transfection of which results in a small amount of knockdownfor both strands. This reduction in signal (˜20%) results in an apparentincrease in reporter protein for all normalized results since thenormalization is based on a negative value of the nonsense siRNA. Thisapparent increase is most noticeable for the passenger strand data ofmodification targets E, F, G, H, J, K, M and O. This model reportersystem is useful to detect changes in the activity of the passengerstrand and the guide strands.

The data of FIG. 4A demonstrate that guide (antisense) strands of siRNAshaving modification Formats E, F, H, K, M and O achieved essentially thesame silencing activity as the control Format A guide strands. In thepresence of modification Formats G and J, the ability of the guidestrands of siRNAs to silence their target was decreased and, in the caseof Format J, the ability of the guide strands of siRNAs to silence theirtarget was reduced to less than 20% knockdown activity on average.

While the majority of modification formats depicted by FIG. 4A providedsimilar guide (antisense) strand mediated cleavage as the unmodifiedcontrol guide strands, all test modification formats severely impairedthe ability of the passenger strands to silence their target essentiallyrendering the passenger strands inactive for interference activity.

To further examine the effects of modification formats on strandactivity, the set of siRNAs used for the strandedness assay was expandedto include a further 18 siRNAs. The data of FIG. 4B-FIG. 4E provide theresults of assays of the larger siRNA collection in box plot formats,also referred to as box and whisker plots. For each plot, the darkhorizontal bar represents the median value of the dataset for eachmodification format. The box represents the distribution of the data atthe lower quartile and the upper quartile of the dataset for eachmodification format while the dotted lines (whiskers) represent thesmallest and largest values of the dataset for each modification format.The circles represent likely outliers in the dataset for eachmodification format.

The activity of the passenger strand following transfection in Helacells of siRNAs of this larger siRNA set is provided by FIG. 4B.Activity is calculated using the following formula: P (passenger strandactivity)=(fLUC remaining from reverse clone treated with test siRNAβ-gal activity)/(fLUC remaining from reverse clone treated with Negcontrol siRNA β-gal activity). The data of FIG. 4B demonstrate a loss ofactivity of the passenger strands of modified siRNA formats as seen bythe upward shift of the median passenger strand knockdown activity whencompared to that of Format A. The data for modification formats G, H, J,K, M and O were statistically significant (p<0.05), therebydemonstrating that the modification formats were efficient at reducingthe activity of the passenger strand.

The activity of the guide strand following transfection in Hela cells ofthe set of siRNAs analyzed for FIG. 4B is provided by FIG. 4C. Activityis calculated using the following formula: G (guide strandactivity)=(fLUC remaining from forward clone treated with test siRNAβ-gal activity)/(fLUC remaining from forward clone treated with Negcontrol siRNA/β-gal activity). Formats E, F, H, M, and O did notnegatively impact the activity of the guide strand (i.e., “do no harm”).Format E appears to increase the ability of the guide strand to reducethe luciferase signal in a statistically significant manner indicated bythe downward shift in the median guide strand activity compared to theunmodified Format A. Modification formats G, J and K reduced theactivity of the guide strand as evidenced by the upward shift in themedian values for those formats (p<0.05) compared to the unmodifiedsiRNAs of Format A.

The data of FIG. 4D provide the difference of fLUC activity between thepassenger and the guide strands of FIG. 4B and FIG. 4C using thefollowing formula: Activity Difference=P−G (using the definitions of Pand G as set forth above). Since the fLUC reporter assay measures theamount of fLuciferase activity following siRNA transfection in cells,more active siRNA strands knock down more of the respective fLUCreporter mRNA and result in lower relative amount of luciferaseactivity. Thus, if the passenger strand is less active than the guidestrand, the values of this activity difference calculation will begreater than 0. If the passenger strand is more active than the guidestrand, the values will be less than 0. Modification Format J reducedthe activity difference between the two strands of the test siRNAs.

The data of FIG. 4E provide the fold change of activity between thepassenger strand and the guide strand for siRNAs of FIG. 4B and FIG. 4C.The activity fold change was calculated as log₂(P)−log₂(G) using thedefinitions of P and G as set forth above. A value of 0 describes asiRNA for which each strand has equal silencing capability. The largerthe activity fold change, the greater the guide strand bias of thesiRNA. A comparison of the box plots of FIG. 4E demonstrates that thestrand bias between the passenger strand and the guide strand issignificantly enhanced for modification formats E, H, M and O (p<0.05)when compared to that of the control unmodified siRNA Format A. Thus,siRNAs having modification Formats E, H, M and O displayed increasedstrandedness as compared to the unmodified Format A. Formats G, J and Kdemonstrated a lower fold difference between passenger and guide strand,but only Format J significantly reduced the fold change of activitycompared to Format A. The largest individual strand bias wasdemonstrated by Format H.

A study was carried out to test the ability of the modification formatsto affect the silencing activity of highly potent siRNA strands bystrand-switching (SW) the modification formats from passenger strand toguide strand and from guide strand to passenger strand for eachmodification format. That is, for modification Format F_(SW) of FIG. 5A,for example, the modifications of Format F at positions 1, 20, and 21 ofthe passenger (sense) strand (FIG. 1A) were introduced into the guide(antisense) strand, and the modifications of Format F at positions 20and 21 of the guide (antisense) strand (FIG. 1A) were introduced intothe passenger (sense) strand.

The data of FIG. 5A provide results of a strand assay where the amountof normalized reporter protein present after carrying out RNAi studiesis plotted for 12 siRNAs having unmodified siRNA Format A and the same12 siRNAs having the modifications of the strands switched as comparedto those of FIG. 4A. The set of siRNAs studied for the FIG. 5A datadiffers from the set studied for the FIG. 4A data in that the siRNAs forthe FIG. 5A data have a strand bias as evidenced by the large differencein the % of fLUC signal remaining between the guide and passenger strandof the unformatted Format A siRNA. The experimental conditions that wereused to measure knockdown in this “strand-switching” assay were the sameas those used to generate the data of FIG. 4A.

As provided by the data of FIG. 5A, the silencing activity of the guidestrands was dramatically reduced due to the strand-switchedmodifications. On average, the guide strands having strand-switchedmodification formats achieved knockdown by less than 20%.Strand-switched modification Format H_(SW) was one of the most potentmodification formats to inactivate the guide (antisense) strand for thiscollection of siRNAs.

The test passenger strands having strand-switched modification formatsprovided variable results. However, all test passenger strands havingstrand-switched modification formats were more effective at knockdownthan in the unmodified siRNAs of Format A in FIG. 5A. While not wantingto be bound by theory, it is possible that, as a result of impairing thepotency of the guide strand, the potency of the passenger strands isindirectly or secondarily enhanced as suggested by the enhancedknockdown achieved by the passenger strands in the strand-switchedmodified formats.

FIG. 5B provides the data of FIG. 5A in a box plot format, which formatis described above as for FIG. 4D. Thus, if the activity of thepassenger strand is less than the activity of the guide strand, thedifference would be expected to be greater than 0, which is demonstratedby modification Format A, for which the median activity difference isapproximately 0.65 for this collection of siRNA. The strand-switchedmodification formats have difference activities less than 0 indicatingthat Formats H_(sw), M_(sw), F_(sw), E_(sw) and O_(sw) switched thestrandedness to favor the passenger strand rather than the guide strandcompared to the unmodified Format A. The greatest enhancement ofstrandedness was observed for strand-switched modification Format M_(sw)which has a median activity difference of ˜−0.5.

The fold change of activity (log₂(P)−log₂(G)) between the passengerstrand and the guide strand for the siRNAs of FIG. 5B is provided foreach modification format in FIG. 5C. These results indicate that amodification format that confers knockdown activity to a guide strandand essentially inactivates a passenger strand, when strand-switched,can reduce knockdown activity of a guide strand and increase knockdownactivity of a passenger strand.

A study similar to that of FIG. 4A and FIG. 4D (using LNA® modifiednucleotides) was carried out using various modification formats wherethe modified nucleotides were 2′-O methylated. For this study, twosiRNAs for each of three target mRNAs were synthesized in the unmodifiedFormat A and in each test format. Therefore, each test format providesdata for 6 siRNAs. As for the studies of FIG. 4A, the particular siRNAsequences chosen for this study are those for which both strands of theunmodified Format A siRNA demonstrated silencing. The guide (antisense)strand of unmodified siRNAs, on average, reduced the target mRNA by 70%while the passenger (sense) strand reduced the target mRNA by 55%.

With regard to passenger (sense) strand activity, the data of FIG. 6Ademonstrate that essentially all mRNA product is available for theluciferase assay after 48 hours incubation with the siRNA in culture.That is, the test siRNAs having modification formats where the modifiednucleotides are 2′-O methylated demonstrate significant loss of potencyof the passenger (sense) strand as indicated by the increase in the %remaining gene expression in these samples. This passenger strandinactivity for knockdown is consistent with the data for themodification formats using LNA® modified nucleotides. Also consistentwith the data for the LNA® modification Formats H, K, and O, the potencyof the guide strands having modification Formats H, K, and O with2′-O-methyl nucleotides was essentially unchanged as compared tounmodified control Format A. Thus, the modification formats with2′-O-methyl modified nucleotides appear to be as effective inmaintaining the activity of the guide (antisense) strand while disablingthe cleavage activity of the passenger (sense) strand in these strandassays for which the target is exogenously-provided.

FIG. 6B provides the data of the siRNAs studied for FIG. 6A (6 siRNAs)plus an additional 18 siRNA sequences in a box plot format. The activitydifference of the Format A group indicates that the majority of thesiRNAs are biased towards the guide strand (indicated by the medianvalue located at about 0.5). However, siRNAs generated with 2′OMe in themodification formats indicated demonstrate a larger difference betweenthe passenger strand and the guide strand as compared to the controlFormat A. Formats H and H+1 induced the largest difference between thestrands. Format O with 2′-OMe modified bases failed to increase theactivity difference between the strands.

The activity fold change of the passenger strand compared to the guidestrand is provided in FIG. 6C, calculated as previously described. Thefold change analysis indicated that Format H provides the largest foldchange compared to unmodified siRNAs of Format A. However, significantfold changes in activity between the strands was also observed byFormats H−1, H, H+1, V−2 and K, but not by Format O as determined by theWilcoxon values cited in the figure legend for FIG. 6C.

A third type of modified nucleotide was studied in various modificationformats. FIG. 7A provides results of strand assays in which modifiednucleotides of modification formats were 2′, 5′-linked nucleotides. Theactivity difference of the fLuc signal from pMIR-REPORT™ studies of GOIcloned in the forward and reverse orientation, thereby representing thedifference in guide and passenger strand efficacy and knockdownactivity, is plotted for unmodified Format A and test Formats H−1, H,H+1, V−2, K and O. A comparison of the strandedness results for theunmodified control Format A to the results for the test formatscontaining 2′,5′-linked bases indicated that no statisticallysignificant changes in the strandedness of the siRNA were obtained byincluding 2′, 5′-linked base modifications with the exception of FormatO. Format O with 2′-5′-linked base modifications decreased the activitydifference indicating a loss of knockdown activity for the guide strandin a statistically significant manner.

The activity fold change between the passenger strand and the guidestrand is provided by FIG. 7B calculated as previously described. Thefold change analysis indicated that Format O containing the 2′-5′ linkednucleic acids influenced the strandedness of the siRNA in a negativedirection as indicated by the decrease in the fold change of activitybetween the passenger strand and the guide strand. Further, data fromthe 2′-5′ linked nucleic acids in formats H−1, H+1, V−2, and K are notstatistically significant and therefore, these formats are not suitableto improve the strandedness of the siRNAs. Only Format H modified with2′-5′ base modifications demonstrated a statistically significantincrease in strandedness as demonstrated by the Wilcoxon values cited inthe figure legend for FIG. 7B.

A statistical analysis on the difference in cleavage activity of theguide versus the passenger strand demonstrated that the greatest changein strand bias was introduced by the LNA® and 2′OMe modified nucleotidesin modification Format H based on the data of FIG. 4A and FIG. 6A. Thestrandedness analysis also demonstrated that Format G, Format J andFormat K decreased the activity of the guide strand in a statisticallysignificant manner, thus they were dropped from subsequent considerationfor enhancing siRNA specificity. Format H with LNA® modified nucleotidesinduced on average an 8-fold difference in cleavage activity of theguide strand over the passenger strand as evidenced by the data of FIG.4A. By comparison, the unmodified control Format A demonstrates, onaverage, a 4-fold increased activity of the guide strand over thepassenger strand. Thus, the Format H modification doubled the strandbias that normally exists in an optimally designed siRNA.

In addition, these data demonstrated that the strand-switched formatshaving LNA® modified residues were capable of inactivating the guidestrand as efficiently as they inactivated the passenger strand.

The data for FIG. 12A-1 and FIG. 12A-2 represent knockdown activity of12 siRNAs generated to contain LNA® modified nucleotides in theindicated formats. The strandedness of the test group indicates that themajority of the siRNAs are asymmetrically biased toward the higheractivity guide strand (indicated by the median difference of activity of0.5 for Format A of FIG. 12A-1). However, siRNAs generated with themodification formats and LNA® residues demonstrated a larger differencebetween the passenger strand and the guide strand activity as comparedto unmodified Format A. The Format H modification format enhanced theactivity difference between the strands in a statistically significantmanner, but the fold change of activity was not significant as shown bythe data for FIG. 12A-2.

The data for FIG. 13A-1 and FIG. 13A-2 represent siRNAs generated tocontain 2′ OMe modified nucleotides in the indicated formats. Thestrandedness of the test group indicates that the majority of the siRNAsare asymmetrically biased toward the higher activity guide strand asevidenced by the median difference of activity of 0.5 of FIG. 13A-1.However, siRNAs generated with 2′OMe modified nucleotides demonstrate alarger fold change between the passenger strand and the guide strand ascompared to the unmodified control Format A as shown by FIG. 13A-2.Formats H, K, and V−2 induced the largest and statistically significantfold change between the strands (p<0.05). Data for all formats, with theexception of Format O, were statistically significant as compared to theunmodified siRNA.

Example 4 Effects of siRNA Modification Formats on Silencing ofEndogenous Genes

Silencing of endogenous genes by modification formatted siRNA was testedto determine if the modification formats altered the efficacy of thesiRNA to knock down its mRNA target. For this study, knockdown ofendogenous genes was tested by transfection of modification-formattedsiRNAs at low concentration into Hela cells (ATCC No. CCL-2, Manassas,Va.), U2OS cells (a human osteosarcoma cell line, ATCC No. HTB-96), andHUH7 cells (a human hepatoma cell line, # JCRB 0403, Japanese CancerResearch Resource Bank, Tokyo, Japan). The Hela cell data are discussedherein.

Hela cells were transfected using the siPORT™ NeoFX™ Transfection Agent(Ambion/Applied Biosystems Austin, Tex. Cat #AM4510) using the “reverse”transfection protocol described by the manufacturer. Unmodified controlor modification-formatted siRNA was plated into 96-well tissue cultureplates to achieve a final concentration of either 1 nM or 5 nM. SiPORT™NeoFX™ transfection agent was complexed with siRNA for at least 10minutes, followed by overlaying freshly trypsinized Hela cells (˜4.0×10³cells) on the siRNA/transfection agent complex. The plates wereincubated under tissue culture conditions at 37° C. for 48 hours.

U2OS cells were transfected using HyPerFect® Transfection Agent (Qiagen,Gaithersberg, Md.) using the “forward” transfection protocol describedby the manufacturer. Unmodified control or modification-formatted siRNAwas plated into 96-well tissue culture plates to achieve a finalconcentration of either 3 nM or 30 nM. HyPerFect® transfection agent wascomplexed with siRNA for at least 10 minutes and the complex was addedto U2OS cells in culture (about 4.0×10³). The plates were incubatedunder tissue culture conditions at 37° C. for 48 hours.

After harvesting transfected cells, total RNA was isolated using theMagMAX™-96 total RNA isolation kit (Ambion, Inc. Austin Tex. Cat.#AM1830) according to the manufacturer's protocol. RNA was eluted in 50μl of nuclease-free water. cDNA was generated using the High CapacitycDNA Reverse Transcription Kit (Applied Biosystems, Inc. Foster CityCalif., Cat. #4368814) according to the manufacturer's protocol in a 30μl reaction volume. TaqMan® Gene Expression Assays (Applied Biosystems,Inc., Cat. #4331182) were carried out using 2 μl of cDNA in a 10 μltotal PCR reaction volume. Technical replicates were generated and an18S value was calculated for each sample. Preamplification uniformitywas checked by calculating the ddCT using the formula described above.

The % remaining was calculated using the formula: %remaining=100×2^(−(ddCT)).

The fraction of mRNA knockdown was calculated using the followingformula:fraction of mRNA knockdown=1−(2^(−(ddCT)))and% mRNA knockdown=fraction of mRNA knockdown*100.

For this knockdown study, six siRNAs for each of eight endogenous targetmRNAs were synthesized in unmodified Format A and for each of the 8 testmodification Formats E, F, G, H, J, K, M and O. Therefore, each testformat provides data for 48 siRNAs. For the data of FIG. 8 the modifiednucleotides “m” at positions as indicated by the format letter andreferring to FIG. 1A-FIG. 1C were LNA® residues having structure (a)above where R1 is O, R2 is O, and R3 is CH₂.

FIG. 8 provides data for silencing the endogenous mRNA targets in Helacells at 5 nM final concentration of modification-formatted siRNA andassaying the % knockdown of the target 48 hours after transfection usingTaqMan® Gene Expression Assays. The results from these studiesdemonstrate that the modification-formatted LNA®-containing siRNAs inFormats G, J and K compromise the knockdown activity of the siRNA, whilethe modification-formatted LNA®-containing siRNAs in Formats E, F, H, Mand O maintain or increase knockdown compared to that observed for theunmodified Format A group. Thus, several modification formats havingLNA® residues in various positions did not negatively impact theknockdown activity of the siRNA but were sufficient to enhance thestrandedness of the guide strand vs. the passenger strand. The knockdownactivity of the siRNAs in this collection of LNA® modified siRNAs wasalso determined in human hepatoma cells (HUH7), and human osteosarcomacells (U2OS). In these cell lines, the LNA® modified siRNA of eachmodification format displayed similar knockdown activity to thatobserved in HeLa cells taking into account different transfectionefficiencies (data not shown). Therefore, silencing performance was notaffected by the type of cell line used.

Further knockdown activity data are provided by FIG. 12B formodification Formats H, H−2, H−1, H+1, Q, K, V, V−1, and V−2 using LNA®modified nucleotides. This dataset represents 24 different siRNAstargeting 4 different endogenous mRNA targets. Each of the 24 siRNAs wassynthesized in each format and the knockdown of the siRNAs in each groupwas analyzed identically. Transfection was into Hela cells at 5 nM finalconcentration with the assays carried out 48 hours after transfectionusing TaqMan® Gene Expression Assay. The results demonstrate a slightdecrease in the knockdown activity of the siRNAs in formats K and V−2that is not statistically significant from the unmodified siRNA.Modification formats H, H−1, H−2, H+1, Q, V and V−1 maintained orincreased the knockdown activity of mRNA targets when compared to thatobserved for the unmodified siRNA group.

The data of FIG. 13B provide knockdown activity for modification FormatsH, H+1, H−1, K, V−2, and O using 2′OMe modified nucleotides. Thisdataset represents 24 different siRNAs targeting 4 different endogenousmRNA targets. Each of the 24 siRNAs was synthesized in each format andthe knockdown of the siRNAs in each group was analyzed identically.Transfection was into Hela cells at 5 nM final concentration with theassays carried out 48 hours after transfection using TaqMan® GeneExpression Assay. The results demonstrate that modification formats H,H+1, H−1, K, V−2, and O maintain or increase knockdown as compared tothat observed for the unmodified Format A siRNAs.

Further data are provided by FIG. 14A for modification Formats H, M, W,W+1, W−1, Y, Y+1, and Y−1 using LNA® modified nucleotides compared toFormat A. This dataset represents 15 different siRNAs targeting 5different endogenous mRNA targets. Each of the 15 siRNAs was synthesizedin the indicated formats and the knockdown of the siRNA in each groupwas analyzed identically. Transfection was into U2OS cells at a finalconcentration of 30 nM with the assays carried out 48 hours aftertransfection using the TaqMan® Gene Expression Assay. The resultsdemonstrate that siRNAs having modification Formats W, W+1, W−1, Y, Y+1,and Y−1 modified with LNA® residues maintain knockdown activity whencompared to that of unmodified siRNA Format A. SiRNAs havingmodification Format M are compromised in knockdown activity whencompared to that of siRNAs having unmodified Format A, and siRNAs havingmodification Format H improve knockdown activity when compared to thatof unmodified Format A, both in a statistically significant manner.

Data of FIG. 14B provide knockdown activity for modification Formats H,M, JB1, JB2, JB3, JB4, and JB5 using LNA® modified nucleotides comparedto Format A. This dataset represents 15 different siRNAs targeting 5different endogenous mRNA targets. Each of the 15 siRNAs was synthesizedin the indicated formats and the knockdown of the siRNA in each groupwas analyzed identically. Transfection was into U2OS cells at a finalconcentration of 30 nM with the assays carried out 48 hours aftertransfection using TaqMan® Gene Expression Assay. The resultsdemonstrate that siRNAs having modification Formats JB2, JB3, and JB5are not statistically significantly different than unmodified Format Ain terms of knockdown activity; siRNAs having Format H, JB1, and JB4improve knockdown activity when compared to Format A and siRNAs havingFormat M have less knockdown activity when compared to Format A.

Of the siRNA modification formats examined in this example, Formats G,J, K and M compromise knockdown activity when compared to the unmodifiedformat.

Example 5 Effects of siRNA Having Modification Format H on Global GeneExpression Profiling as a Measure of Off-Target Effects

The ability of the LNA® and 2′-O-methyl modified nucleotides in Format Hto influence the signature of differentially expressed genes wascompared to that of unmodified siRNA by measuring the gene profile usinga microarray analysis. Microarray genome analysis was carried out onquadruplicate biological samples transfected with 4 siRNAs designed totarget the FDFT1 gene. FDFT1 is a gene that is involved in cholesterolbiosynthesis and not essential to the cells that are used for thetesting. This criterion ensures that the differences in gene expressionthat are detected by array analysis are solely the results of cleavagemediated by the siRNA rather than a result of any biological cascadesthat result from the loss of the functional mRNA target.

Each of three siRNA sequences (designated siRNA #183, #184 and #192) wasseparately transfected in the unmodified Format A, LNA® modified FormatH and 2′-O-methyl modified Format H into Hela cells using standardprocedures as outlined above and transfected cells were harvested 24hours later. RNA was isolated using standard procedures for microarrayanalysis. Affymetrix U133 V2 microarray chips (Affymetrix, Santa Clara,Calif.) containing human probesets were used for these studies. Controlsincluded a negative control siRNA, “mock” transfected cells, andnontreated cells to record baseline levels of gene expression in theabsence of experimental treatment. Following normalization of the data,the results were analyzed to determine the identity of the genes thatwere changed by 2-fold or greater in each of the sample sets as comparedto the mock (delivery agent only) control sample results. A 1-way ANOVAwith planned contrasts was then used to determine the p-values for eachmock-treated vs. experimental condition. The differentially expressedgene probe sets were then defined as 2-fold changes with p<0.001. Thenumber of differentially expressed genes is a measure of off-targeteffects due to the siRNA.

FIG. 9A-FIG. 9C provide Venn diagrams of the results for siRNA #183(FIG. 9A), siRNA #184 (FIG. 9B) and siRNA #192 (FIG. 9C), three distinctsiRNAs designed to target the FDFT1 gene. Each diagram depicts resultsfor one of three siRNAs in unmodified, 2′-O-methyl or LNA® modifiedsiRNAs as indicated. The Venn diagrams indicate the number of specificgenes that were changed in each experimental condition and theintersection of the genes between siRNA conditions. For each diagram,the lower left set (red if in color) indicates the number of specificgenes changed 2-fold or greater in the unmodified siRNA treated samples,the upper set (blue if in color) indicates the number of specific geneschanged 2-fold or greater in the 2′-O-methyl Format H modified siRNAtreated samples, and the lower right set (green if in color) indicatesthe number of specific genes changed 2-fold or greater in the LNA®Format H modified siRNA treated samples.

For siRNA #183 (FIG. 9A), the unmodified siRNA induced a 2-fold orgreater decrease in the mRNA levels of 31 genes (2+19+10+0). The2′-O-methyl modified siRNA induced a 2-fold or greater decrease in themRNA levels of 32 genes (3+0+10+19), 29 of which were also affected bythe unmodified control siRNA. Samples treated with 2′-O-methyl modifiedsiRNA differed from the unmodified siRNA in the number of off-targetgenes by only 3 genes. The LNA® modified siRNA induced a 2-fold orgreater decrease in the mRNA levels of 10 genes (0+0+10+0), all of whichare also altered by the unmodified control and the 2′-O-methylmodification. Therefore, those 10 genes represent off-target effectsthat are sequence-dependent and cannot be alleviated by the teachingsprovided herein. Thus, the LNA® modification format had the moresignificant impact on the removal of off-target gene expression changes(a 68% decrease in off-target effects) as compared to the 2′-O-methylmodification (a 0% decrease in off-target effects).

For siRNA #184 (FIG. 9B), the unmodified siRNA induced a 2-fold orgreater decrease in the mRNA levels of 70 differentially expressed genesin Hela cells (14+23+33+1). The 2′-O-methyl modified siRNA induced a2-fold or greater decrease in the mRNA levels of 66 genes (9+1+33+23).In contrast, the LNA® modified siRNA reduced the number ofdifferentially expressed genes by 50% (35 genes (0+1+33+1)) compared tothe unmodified siRNA results. The vast majority of the differentiallyexpressed genes in the LNA® modified treated samples (33 genes) werealso identified as differentially expressed in unmodified and2′-O-methyl modified siRNA treated samples. Such genes representoff-target effects that are sequence-dependent and cannot be alleviatedby the teachings provided herein. Thus, the LNA® modification formatagain had the more significant impact on the removal of off-target geneexpression changes (a 50% decrease in off-target effects) as compared tothe 2′-O-methyl modification (a 6% decrease in off-target effects).

For siRNA #192 (FIG. 9C), the unmodified siRNA induced a 2-fold orgreater decrease in the mRNA levels of 103 differentially expressedgenes (28+13+61+1). The 2′-O-methyl modified siRNA induced a 2-fold orgreater decrease in the mRNA levels of 81 genes (7+0+61+13). The LNA®modified siRNA induced a 2-fold or greater decrease in the mRNA levelsof 64 genes (2+1+61+0). Thus, the LNA® modification removed ˜38% of theoff-target effects compared to the unmodified siRNA. For this siRNA, 61genes represent off target effects that are sequence-dependent andcannot be alleviated by the teachings provided herein. The LNA®modification format again had the more significant impact on the removalof off-target gene expression changes (a 38% decrease in off-targeteffects) as compared to the 2′-O-methyl modification (a 21% decrease inoff-target effects).

In summary, the microarray analyses demonstrated that LNA® modifiedFormat H siRNA produced 38% to 68% fewer differentially expressed geneswhen compared to unmodified Format A siRNA and the 2′-O-methyl modifiedFormat H siRNA produced 0% to 22% fewer differentially expressed geneswhen compared to unmodified Format A siRNA. Therefore, the LNA® modifiedFormat H siRNA provided the least number of off-target effects.

Example 6 Performance of Modification-Formatted siRNAs in Cell BiologyStudies

Cell biology studies were carried out to gauge the ability of themodification formats and the types of modified nucleotides to impact theon-target and the off-target phenotypes evoked by the siRNAs.

In order to determine if modification-formatted siRNAs maintain the“on-target” or desired phenotype and alleviate the “off-target” orundesired phenotype, siRNAs were chosen that induced a measurable andhighly characterized biological response. Thus, the preservation of theon-target phenotype was monitored in addition to removal of theoff-target phenotype. SiRNAs were synthesized with the test modificationformats and transfected into the appropriate cells for assays asdescribed below.

An assay, termed herein a “growth assay,” was carried out to investigatethe differences in modification-formatted siRNAs compared to unmodifiedsiRNA and negative control siRNA at the cellular level. The growth assayincluded measuring cell phenotype-related parameters such asproliferation, apoptosis and morphologies in an U2OS osteosarcoma cellline following siRNA transfection at 30 nM and at 3 nM. The results fromthe 30 nM assays are discussed herein since more off-target effects areobserved at the higher concentration of siRNA and, therefore, thebarrier for removing such off-target effects is higher. The growth assayanalysis included immunofluorescence detection of the following antigenswhich are markers for the indicated phenotype:

-   -   1. Cleaved lamin-A (apoptosis),    -   2. Phosphorylated Histone H3 (mitosis),    -   3. Tubulin (cytoskeletal morphology which is a quality control        measurement, i.e., a background determination for the structure        of the cell and which marks all cells), and    -   4. Hoescht stain (nuclear morphology which provides cell number        normalization).

Immunofluorescence was collected using a 10× objective powered byrobotically controlled image acquisition software (Cellinger, Munich,Germany) and the METAMORPH® image analysis software package (MolecularDevices, Toronto, Canada) using the IMAGEXPRESS^(Micro)™ automatedfluorescence microscope (Molecular Devices, Toronto, Canada). Fourpositions in each 384 well sample were collected, the data were averagedacross three biological replicates and compared to negative siRNAcontrol samples (scrambled non-targeting siRNA) treated in a similarmanner. Following image analysis, data are evaluated according to thefollowing protocol:

-   -   1) Index calculation: for each site within a well, the number of        mitotic nuclei and apoptotic nuclei was normalized against the        total number of nuclei and the area of non-cell background was        subtracted from the total image area to calculate the area        occupied by cells, and then normalized against the number of        nuclei;    -   2) Sample well average calculation: the average of all sites        within a well was calculated and, additionally, all sites of all        negative control wells per plate were averaged;    -   3) Normalization: samples and positive controls for each plate        of a triplicate were normalized against the average of negative        siRNA control wells;    -   4) Triplicate average calculation: the average of the plate        triplicates was calculated for each treatment and readout.

Test genes were chosen that are well characterized functionally invarious cells by published literature. The test genes included those ofTable 1.

TABLE 1 Expected on-target phenotype mRNA target due to knockdown of theGene Gene gene product Gene description symbol ID Apoptosis Mitosis BUB1budding BUB1B 701 Neutral Decrease uninhibited by benzimidazoles 1homolog beta WEE1 homolog WEE1 7465 Increase Increase (S. pombe) Celldivision cycle 2 CDC2 983 Neutral Decrease Cyclin-dependent CDK2 1017Neutral Decrease kinase 2 Cyclin D1 CCND1 595 Neutral Decrease Polo-likekinase 1 PLK1 5347 Increase Increase Caspase 3 CASP3 836 Neutral NeutralFarnesyl-diphosphate FDFT1 2222 Neutral Neutral farnesyltransferase 1Low Density LDLR 3949 Neutral Neutral lipoprotein receptorSterol-C5-desaturase SC5DL 6309 Neutral Neutral (ERG3 delta-5-desaturase homolog, S. cerevisiae)-like

SiRNAs were identified that provided a strong measurable induction of anexpected phenotype, e.g., a 300% increase in mitosis following knockdownof Weel or PLK1 (Watanabe, N. et al. (2004) PNAS, 101, 4419-4424; Leach,S. D. et al. (1998) Cancer Research, 58, 3132-3136; Cogswell, J. P. etal. (2000) Cell Growth and Diff. 11, 615-623). Knockdown of the BUB1Bgene is expected to decrease the % of mitosis compared to Neg control(Lampson, M. A. and Kapoor, T. M. (2004) Nature Cell Biology, 7, 93-98).

Additionally, siRNAs were identified that displayed an off-targetphenotype. For example, siRNAs to LDLR, a gene not expected to induceapoptosis (see Table 1 for other such genes), demonstrated a 500%increase in apoptosis. Such siRNA sequences served as tools to determineif modification formats could improve the specificity of the siRNA.Separate sets of siRNAs with observable on-target phenotypes andoff-target phenotypes were generated to contain several modificationformats, and either 2′-O-methyl modified nucleotides or LNA® residueshaving structure (a) above where R1 is O, R2 is O, and R3 is CH₂. Thesemodified siRNAs were tested for the on-target and the off-targetphenotypes to determine those modification formats that could remove theoff-target effects while maintaining the on-target phenotype.

The modification-formatted siRNA cell-based assay results are plotted asbox and whiskers plots in FIG. 10A-FIG. 10E, FIG. 11A-FIG. 11F, FIG.12C-FIG. 12D, FIG. 13C-FIG. 13D, FIG. 15A-15D, and FIG. 16A-FIG. 16Drelative to the normalized values for the negative control siRNA treatedsamples analyzed in parallel in the cell-based assays (labeled as Neg insaid figures).

A box plot of normalized mitotic cells resulting from silencing by siRNAhaving LNA® modification Formats E, F, G, H, J, K, M and O is providedby FIG. 10A. The siRNAs targeted BUB1B, WEE1, CDC2, CDK2, CASP3, andCCND1, genes that include disparate effects on mitosis phenotype. Thatis, this set of genes includes a subset that increases mitosis whensilenced, a subset that decreases mitosis when silenced, and a subsetthat is expected to have no effect on mitosis when silenced. Because ofthese disparate effects, a subset of the genes, knockdown of whichprovides the expected phenotype of a decrease in mitosis, was analyzedand the data provided in FIG. 10B.

The FIG. 10B data show normalized mitotic cells following transfectionof siRNAs targeting genes, knockdown of which is expected to decreasemitosis (BUB1B, CDC2, CCND1) (Lampson, M. A. and Kapoor, T. M. (2004)Nature Cell Biology, 7, 93-98; Harborth, J. et al. (2001) Journal ofCell Science 114, 4557-4565; Klier, M. (2008) Leukemia, EPUB). Thesedata therefore measure an on-target phenotypic effect, that is, thisassay ensures that the modification formatted siRNAs “do no harm” to afunctioning set of siRNAs. The normalized values for Neg control treatedsamples show a median value of about 1.0. The unmodified Format A siRNAsprovided a median value of about 0.55 indicating an about 2-folddecrease in mitosis. All modification formats provided for a mitosisdecrease as compared to Neg control. SiRNAs having modification FormatsH and J provided a statistically significant difference in their effecton mitosis (less on-target effect) as compared to unmodified Format A asevidenced by the Wilcoxon values and the shift away from unmodifiedFormat A by the median value for mitosis. Thus, consistent with theresults regarding mRNA knockdown of FIG. 8, cell-based assaysdemonstrate that Format J is not compatible with the desiredcharacteristics of a highly functional siRNA.

The cell-based growth assay also measured apoptosis compared to Negcontrol treated samples by quantitating the fluorescent signal fromcells displaying cleaved lamin-A (Rao, L. et al. (1996) J Cell Biology,135, 1441-1455). Historically, the most prevalent unexpected phenotypeobserved in genome-wide siRNA screening experiments is cell death. Inthis collection of siRNA for these studies, siRNA sequences wereincluded that were known to induce unexpected cell death as a tool todetermine if the modification format could remove the unwanted cellularphenotype.

The results of quantitating the apoptotic fragments due to treating U2OScells with siRNA having LNA® modification Formats E, F, G, H, J, K, Mand O are provided by FIG. 10C. Neg control siRNA treated samples wereused to establish a baseline of apoptosis following transfection ofsiRNA and the data were normalized to 1.0. The siRNA in this collectiontarget the following genes: BUB1B, CDC2, CCND1, WEE1 CASP3, and CDK2, aset of gene targets, the knockdown of a subset of which is expected toincrease apoptosis and a subset of which is expected to have no effecton apoptosis. Because of these disparate effects, a subset of the genes,knockdown of which provides the expected phenotype of an increase inapoptosis, was analyzed and the data provided in FIG. 10D.

FIG. 10D provides the results of apoptosis measurements in the U2OScells following transfection of siRNA targeting WEED. Knockdown of WEE1is expected to increase apoptosis (Watanabe, N. et al. (2004) PNAS, 101,4419-4424; Leach, S. D. et al. (1998) Cancer Research, 58, 3132-3136).These data therefore measure an on-target phenotypic effect, that is,this assay ensures that the modification formatted siRNAs “do no harm”to a functioning set of siRNAs. Neg control siRNA treated samples wereused to establish the normalized baseline of apoptosis. Unmodified siRNAof Format A targeting WEE1 displayed a median of approximately 7.5compared to NEG treated samples, thereby providing the expectedphenotypic change. Modification-formatted siRNA having Formats J and Kreduced the on-target phenotype by reducing the amount of apoptosis in astatistically significant manner when compared to the unmodified FormatA siRNA demonstrating that Formats J and K compromise the performance ofthe siRNA. These data are consistent with those of FIG. 8 regardingknockdown by siRNAs having modification Formats J and K.

Analysis of the ability of modification formatted siRNAs to remove anoff-target phenotype for a subset of genes, knockdown of which isexpected to have no effect on apoptosis, is provided by the data of FIG.10E. SiRNAs that have empirically demonstrated apoptosis off-targeteffects were specifically chosen for study to determine if such siRNAs,when modification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has an increased median and greaterdistribution of data as compared to the Neg control (FIG. 10E). ThesiRNA studied have Formats A, E, F, G, H, J, K, M and O and the targetgenes, knockdown of which is not expected to induce apoptosis, includedBUB1B, CCND1, CDK2, and CDC2. The Neg control treated population wasused to establish the baseline for apoptosis. SiRNA having Formats G, H,J and K demonstrate a statistically significant ability to removeoff-target effects as evidenced by the lower apoptotic signal comparedto the unmodified Format A (FIG. 10E).

In light of the data of FIG. 10D where siRNA having modification FormatsJ and K were demonstrated to compromise the siRNA on-target efficacy andin light of the data of FIG. 8 where siRNA having modification FormatsG, J, and K were demonstrated to compromise knockdown activity, FormatsG, J, and K appear to “do harm” to an otherwise functioning siRNA.

Further characterization of the modification formats was carried out byusing modified nucleotides containing 2′O-methylated residues instead ofLNA® residues to determine if other types of modifications could reduceoff-target effects by siRNAs. FIG. 11A provides a box plot of normalizedmitotic cells resulting from silencing by siRNAs having 2′-O-methylmodification Formats H and K and targeting WEE1, PLK1, FDFT1, LDLR, andSC5DL, genes that include disparate effects on mitosis phenotype. Thatis, this set of genes includes a subset that increases mitosis whensilenced, and a subset that is expected to have no effect on mitosiswhen silenced. Because of these disparate effects, a subset of thegenes, knockdown of which provides the expected phenotype of an increasein mitosis, was analyzed and the data provided in FIG. 11B.

FIG. 11B provides the results of mitosis measurements in U2OS cellsfollowing transfection of siRNA targeting WEE1 or PLK1 by siRNA having2′-O-methyl modification Formats H and K. That silencing of WEE1 or PLK1results in an increase of mitosis in U2OS cells is provided by Watanabe,N. et al. ((2004) PNAS, 101, 4419-4424), Leach, S. D. et al. ((1998)Cancer Research, 58, 3132-3136), and Cogswell, J. P. et al. ((2000) CellGrowth and Diff. 11, 615-623) and confirmed by the data for siRNAshaving unmodified Format A, which induced an about 4-fold increase inmitosis compared to the Neg control treated samples. These datatherefore measure an on-target phenotypic effect, that is, this assayensures that the modification formatted siRNAs “do no harm” to afunctioning set of siRNAs. As shown by the data of FIG. 11B, similarlevels of impact on mitosis are observed using siRNAs having 2′OMemodification Formats H and K. This study demonstrates that the 2′OMemodification in the Formats H and K do no harm to an otherwisefunctioning siRNA.

To determine if siRNAs having 2′ OMe modification Formats H and K couldreduce off-target effects, siRNAs targeting a subset of genes (FDFT1,SC5DL and LDLR), knockdown of which is expected to have no effect onmitosis, were transfected into the U2OS cells and the impact on mitosiswas determined. However, siRNAs that have empirically demonstratedmitosis off-target effects were specifically chosen for study todetermine if such siRNAs, when modification-formatted, would eliminateor reduce the off-target effects. Data are provided by FIG. 11C. Thatthe siRNAs have off-target effects is evidenced by the box plot forunmodified Format A which has a greater distribution of data as comparedto the Neg control (FIG. 11C). SiRNA having 2′OMe residues in Format Hreduced many of the off-target phenotypes observed in the unmodifiedsiRNA Format A when individual siRNA sequences were compared. SiRNAshaving 2′OMe residues in Format K raised the median mitotic impactcompared to the unmodified siRNA. However, data for this 2′-O-methylmodification in Formats H and K were not statistically significantlydifferent from the data of unmodified Format A.

The results of quantitating apoptotic fragments due to treating U2OScells with siRNA having 2′-O-methyl modification Formats H and K areprovided by FIG. 11D. Neg control siRNA treated samples were used toestablish a baseline of apoptosis following transfection of siRNA andthe data were normalized to 1.0. The siRNA in this collection targetgenes WEE1, PLK1, FDFT1, LDLR, and SC5DL, knockdown of a subset of whichis expected to increase apoptosis and a subset of which is expected tohave no effect on apoptosis. Because of these disparate effects, asubset of the genes, knockdown of which provides the expected phenotypeof an increase in apoptosis, was analyzed and the data provided in FIG.11E.

FIG. 11E provides the results of apoptosis measurements in the U2OScells following transfection of siRNA targeting WEE1 and PLK1 by sets of12 siRNAs having 2′-O-methyl modification Formats H and K. These datatherefore measure an on-target phenotypic effect, that is, this assayensures that the modification formatted siRNAs “do no harm” to afunctioning set of siRNAs. Neg control siRNA treated samples were usedto establish the normalized baseline of apoptosis. Unmodified siRNA ofFormat A targeting WEE1 or PLK1 displayed a median of greater than abouta 4-fold increase in apoptosis compared to NEG treated samples, therebyproviding the expected phenotypic change. Similar levels of apoptosiswere observed with 2′OMe modified siRNA in Format H and Format K. Thisstudy demonstrates that 2′OMe modifications in the H and K formats donot reduce the efficacy of the siRNA and provide similar phenotypes asobserved in the unmodified siRNA Format A.

Analysis of the ability of 2′-O-methyl modification formatted siRNAs toremove an off-target phenotype for a set of genes, FDFT1, LDLR, andSC5DL, knockdown of which is expected to have no effect on apoptosis, isprovided by the data of FIG. 11F. SiRNAs that have empiricallydemonstrated apoptosis off-target effects were specifically chosen forstudy to determine if such siRNAs, when modification-formatted, wouldeliminate or reduce the off-target effects. The siRNA studied haveFormats A, H and K. The Neg control treated population was used toestablish the baseline for apoptosis and has a particularly broad rangeof apoptosis results. The median value for the control Format A wassimilar to that of the Neg control treated samples. SiRNAs having 2′OMemodified nucleotides in modification Formats H and K reduced many of theoff-target phenotypes observed in the unmodified siRNA Format A whenindividual siRNA sequences were evaluated, but over the test population,the reduction of the off-target effects was not observed in astatistically relevant manner.

Further modification formats were tested as set forth in the schematicof FIG. 1A-FIG. 1C that provide Formats H, H−2, H−1, H+1, Q, V, V−1, V−2and K. The LNA® modified nucleotides had structure (a) where R1 and R2are O, R3 is CH2, and R4 and R5 are as determined by the position of thenucleotide in the siRNA as described herein. The on-target phenotypeassay for mitosis (i.e., the “do no harm” assay) in U2OS cells wascarried out for the siRNA modification formats of FIG. 12C. As shown bythe data of FIG. 12C, unmodified siRNA demonstrated an about 2-foldincrease in mitosis in siRNA treated cells compared to Neg controltreated cells. The on-target performance of the modification-formattedsiRNAs modified by LNA® in these formats did not negatively impact theexpected phenotype. Modification Format H provides a statisticallysignificant gain in phenotype.

FIG. 12D provides data regarding removal of off-target effects forsiRNAs having the LNA® modification formats of FIG. 12C. A box plot ofnormalized apoptotic fragments resulting from silencing by siRNA havingunmodified Format A and for LNA® modification Formats H, H+1, H−1, H−2,K, Q, V, V−1, and V−2, for a set of gene targets, the knockdown of whichis expected to have no effect on apoptosis (FDFT1, LDLR or SCSDL). Thenegative control (Neg) is a scrambled non-targeting siRNA for whichapoptosis quantitation is normalized to a value of 1.0. However, siRNAsthat have empirically demonstrated apoptosis off-target effects werespecifically chosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has an increased median and greaterdistribution of data as compared to the Neg control. The modificationformats that were most effective at removing the unwanted apoptosisphenotype were Formats H, K, Q, and V−2 while modification Formats H−2,H−1, H+1, V, and V−1 did not alter the apoptotic fragments measurementwith any statistical significance as compared to unmodified siRNA.

SiRNAs modified by 2′OMe nucleotides for modification formats H, H−1, K,and V also do not negatively impact the on-target phenotype in U2OScell-based assays as shown by the data of FIG. 13C. FIG. 13C provides abox plot of normalized mitotic cells resulting from silencing by siRNAhaving unmodified Format A and for said modification formats having2′-O-methyl modified nucleotides for a set of gene targets, theknockdown of which is expected to increase mitosis (WEE1 and PLK1). Thisassay is a “do no harm” assay. Unmodified siRNA demonstrated a medianvalue for mitosis of ˜2-fold greater than the Neg control treatedsamples as expected. The data for the modification formatted siRNAdemonstrate that siRNA having 2′OMe modified nucleotides do notnegatively impact the expected phenotype in cell-based assays.

FIG. 13D provides a box plot of normalized apoptotic fragments resultingfrom silencing by siRNAs having unmodified Format A and for siRNAshaving modification Formats H, H−1, V and K having 2′-O-methyl modifiednucleotides for a set of gene targets, the knockdown of which isexpected to have no effect on apoptosis (FDFT1, SC5DL or LDLR). However,siRNAs that have empirically demonstrated apoptosis off-target effectswere specifically chosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has greater distribution of data ascompared to the Neg control. The set of siRNAs contained 13 differentsiRNAs for each format. None of the siRNAs having 2′OMe modificationformats provided data that are statistically significantly differentfrom unmodified control Format A demonstrating that the 2′OMemodification formatted siRNA maintained the same off-target effectsobserved for the unmodified Format A siRNA.

Further modification formats similar to that of Format H and as setforth in FIG. 1D were studied. FIG. 15A provides box plots of normalizedmitotic cells for siRNAs having LNA® modified nucleotides in Formats A,H, M, W, W+1, W−1, Y, Y+1, and Y−1. The siRNA set was chosen toknockdown BUB1B, CCND1, and CDC2 mRNAs to result in an expected decreaseof the mitotic index of U2OS cells. These data therefore measure anon-target phenotypic effect, that is, this assay ensures that themodification-formatted siRNAs “do no harm” to a functioning set ofsiRNAs. As provided by the data of FIG. 15A, the median mitotic valuefor samples treated with unmodified Format A siRNA demonstrated adecrease in mitosis to approximately 0.75 compared to the Neg controlsiRNA treated samples. Modification-formatted siRNA in Formats H, M,W+1, W−1, Y−1 and Y+1 do not significantly change the impact of thesesiRNA on mitosis demonstrating that these modification formats “do noharm.” However, modification Formats Y and W reduced the expectedon-target impact of the siRNAs demonstrating that these modificationformats interfere with on-target function.

FIG. 15B provides a box plot as for FIG. 15A for CASP3, knockdown ofwhich is not expected to affect mitosis. However, siRNAs that haveempirically demonstrated mitosis off-target effects were specificallychosen for study to determine if such siRNAs, whenmodification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has a median value of about 0.7 ascompared to the Neg control. SiRNAs having modification Formats H, W,W+1, W−1, Y, and Y+1 reversed the off-target effects demonstrated bysiRNAs of Format A. Modification of siRNA with Formats M and Y−1 did notreverse the off-target phenotypes of the Format A siRNAs. None of theWilcoxon values provided a p<0.05 for the data of FIG. 15B, possibly dueto the small population of siRNAs in each format.

Box plots of normalized apoptotic fragments for a set of siRNAstargeting the WEE1 gene, knockdown of which is expected to increaseapoptosis, for Formats A, H, M, W, W+1, W−1, Y, Y+1, and Y−1 areprovided by FIG. 15C. Unmodified Format A siRNA show a median apoptoticvalue greater than 4.0 compared to Neg control siRNA treated samplesdemonstrating a robust increase in apoptosis as a consequence of WEE1knockdown. The test data therefore measure an on-target phenotypiceffect, that is, this assay ensures that the modification-formattedsiRNAs “do no harm” to a functioning set of unmodified siRNAs. Asprovided by the data of FIG. 15C, siRNA having modification Formats H,M, and W−1 show similar increases in apoptosis as compared to Format A,whereas Formats W, W+1, Y, Y+1 and Y−1 demonstrate a lesser impact onapoptosis compared to the unmodified siRNA Format A. None of theWilcoxon values provided a p<0.05 for the data of FIG. 15C, possibly dueto the small population of siRNAs in each format. Despite the reducedapoptosis impact, the chemical modification Formats W, W+1, Y, Y+1 andY−1 maintain the ability to knock down the target mRNA (FIG. 14A) and toinduce apoptosis compared to the Neg control siRNA treated samples.

Box plots of normalized apoptotic fragments for a set of gene targets,knockdown of which is not expected to impact apoptosis, are provided byFIG. 15D for siRNAs having modification Formats A, H, M, W, W+1, W−1, Y,Y+1, and Y−1. The siRNA in this set target BUB1B, CCND1, CDC2, or CDK2.However, siRNAs that have empirically demonstrated apoptosis off-targeteffects were specifically chosen for study to determine if such siRNAs,when modification-formatted, would eliminate or reduce the off-targeteffects. That the siRNAs have off-target effects is evidenced by the boxplot for unmodified Format A which has a median value of greater than3.5 and greater distribution of data as compared to the Neg control.Silencing by siRNA having Formats H, Y, and Y+1 show statisticallysignificant reversal of off-target effects as compared to Format A.

Even further siRNA modification formats as set forth in FIG. 1E werestudied. The siRNAs were transfected into U2OS cells and the mitosiseffects were measured as described herein. FIG. 16A provides box plotsof normalized mitotic cells for siRNAs having unmodified Format A andfor LNA® modification Formats H, M, JB1, JB2, JB3, JB4 and JB5. ThesiRNA set (3 siRNAs per target mRNA) was chosen to knockdown BUB1B,CCND1, and CDC2 mRNAs to result in an expected decrease of the mitoticindex of U2OS cells. These data therefore measure an on-targetphenotypic effect, that is, this assay ensures that themodification-formatted siRNAs “do no harm” to a functioning set ofsiRNAs. As provided by the data of FIG. 16A, the median mitotic valuefor samples treated with unmodified Format A siRNA demonstrated adecrease in mitosis to approximately 0.75 compared to the Neg controlsiRNA treated samples. Modification-formatted siRNA in Formats H, M,JB1, JB2, JB4 and JB5 do not significantly change the on-target impactof the siRNA on mitosis demonstrating that these formats are toleratedby siRNA. However, siRNA having modification Format JB3 significantlyreduced the expected on-target impact of the siRNAs targeting thesegenes, demonstrating that Format JB3 interferes with on-target function.

FIG. 16B provides a box plot for modification formats as for FIG. 16Afor a gene target, CASP3, knockdown of which is not expected to affectmitosis. However, siRNAs that have empirically demonstrated mitosisoff-target effects were specifically chosen for study to determine ifsuch siRNAs, when modification-formatted, would eliminate or reduce theoff-target effects. That the siRNAs have off-target effects is evidencedby the box plot for unmodified Format A which has a median value ofabout 0.65 thereby displaying a decrease in mitosis as compared to theNeg control. Modification Formats H, JB1, JB2, JB4 and JB5 were able toreverse these off-target effects as demonstrated by the data of FIG.16B. Modification Formats M and JB3 did not reduce the off-targetphenotypes of the siRNAs targeting CASP3, demonstrating that theseformats are not effective at removing off-target phenotypes. None of theWilcoxon values provided a p<0.05 for the data of FIG. 16B, possibly dueto the small population of siRNAs in each format.

Box plots of normalized apoptotic fragments resulting from silencing thegene target, WEE1, the knockdown of which is expected to increaseapoptosis, are provided by FIG. 16C for siRNAs having modificationFormats A, H, M, JB1, JB2, JB3, JB4 and JB5. These data thereforemeasure an on-target phenotypic effect, that is, this assay ensures thatthe modification-formatted siRNAs “do no harm” to a functioning set ofsiRNAs. As provided by the data of FIG. 16C, the median apoptotic valuefor samples treated with unmodified Format A siRNA demonstrated anincrease in apoptosis to >4.0 compared to the Neg control siRNA treatedsamples demonstrating a robust increase in apoptosis as a consequence ofWEE1 knockdown. SiRNAs having the modification formats induced theexpected phenotype to varying degrees demonstrating that all formatstested do no harm to a set of functional siRNA. None of the Wilcoxonvalues provided a p<0.05 for the data of FIG. 15B, possibly due to thesmall population of siRNAs in each format.

FIG. 16D provides a box plot for modification formats as for FIG. 16Cfor the set of gene targets containing BUB1B, CCND1, CDC2, and CDK2,knockdown of which is not expected to have an effect on apoptosis, forsiRNAs having LNA modified nucleotides in Formats A, H, M, JB1, JB2,JB3, JB4 and JB5. However, siRNAs that have empirically demonstratedapoptosis off-target effects were specifically chosen for study todetermine if such siRNAs, when modification-formatted, would eliminateor reduce the off-target effects. That the siRNAs have off-targeteffects is evidenced by the box plot for unmodified Format A which hasan increased median value of greater than 3 and greater distribution ofdata as compared to the Neg control treated samples. As shown by thedata of FIG. 16D, siRNA having modification Formats H, JB1, JB2, JB3,JB4 and JB5 are able to reverse the apoptosis off-target effects whencompared to that of Format A in a statistically significant manner.SiRNAs having Format M are not able to reverse the apoptotic effect to astatistically significant degree.

When taken together, the data of FIG. 10E, 11C, 11F, 12D, 13D, 15B, 15D,16B, and 16D demonstrate that those modification formats that providefor an unexpected reduction of off-target phenotypic effects includeFormats G, H, J, K, Q, V−2, Y, Y+1, JB1, JB2, JB3, JB4, and JB5. Ofthose modification formats, Formats G, J, and K “do harm” to the abilityof otherwise functional siRNA to knock down activity. Highly functionalsiRNAs have the properties of reducing off-target phenotypic effectsand, in addition, “do no harm” in terms of knockdown activity.Modification formats that provide such high functionality to otherwisefunctional siRNAs include Formats H, Q, V−2, Y, Y+1, JB1, JB2, JB3, JB4,and JB5.

Those modification formats that were studied both for strandednesseffect (data of FIG. 4E, 6C, 7B, 12A, 13A, Example 3 herein) and werestudied for their ability to confer a reduction of off-target effects asprovided above provide an assessment of whether one can predict, fromthe strandedness assay, how a modification formatted siRNA will behavein removal of off-target effects. The strandedness data of Example 3using classic reporter assays demonstrate that modification Formats E,G, H, K, O and M would be expected to perform well in cellularphenotypic assays. In fact, three of these formats did perform well andthree of them did not. Therefore, strandedness assays appear to beineffective at predicting whether a modification formatted siRNA will befunctional at achieving a desired cellular phenotype.

Example 7 Stabilized Modification-Formatted siRNAs

The present example provides modification-formatted siRNAs that areparticularly stable to exposure to biological fluids containingnucleases in addition to maintaining the properties of knockdownactivity and removal of off-target effects discussed above. Elmen et al.(Nucleic Acids Research 33:1, 439-447, 2005) reportedly describe astabilized siRNA termed siLNA5 that has the same modification format asFormat F herein and which format is used as a reference control forstability studies for the present example. The term “stable to exposureto biological fluids,” as used herein, means that the siRNAs maintainstheir full-length in the presence of a biological fluid containingnucleases to the same or greater degree as compared to siRNAs havingmodification Format F exposed to the same biological fluid for the sameperiod of time.

While the present example provides stabilized modification formats,modification-formatted siRNAs as provided by previous examples aresuitable for use in circumstances where nuclease activity is of minimalconcern.

Regarding Format F, the data from Example 6 and FIG. 10E demonstratethat siRNAs having modification Format F are substantially ineffectivein removing off-target cellular effects.

Studies were carried out on 8 different siRNA sequences targeting theCLTC and WEE1 mRNAs to determine those modification formats that conferstability to the siRNAs in 90% mouse blood serum. Synthesis of siRNA wascarried out as described in Example 1 for modification formats describedbelow and set forth by FIG. 1F-FIG. 1K.

Modified Positions (Numbered from the 5′ end of each Strand) FormatPassenger Sequence Guide Sequence A, Control None None F (Elmen et al.)1, 20, 21 20, 21 H 1, 2, 13, 14 None DH21 1, 2, 13, 14 20, 21 DH20 1, 2,13, 14 19, 20, 21 DH3 1, 2, 13, 14, 20, 21 None DH30 1, 2, 13, 14, 19,20 None DH35 1, 2, 13, 14, 20 21 DH6 1, 13, 14, 21 21 DH34 1, 2, 13, 14,20, 21 21 DH2 1, 2, 13, 14, 20, 21 20, 21 DH19 1, 2, 13, 14, 19, 20 19,20, 21 DH4 1, 2, 13, 14, 19, 20 19, 20 DH31 1, 2, 13, 14, 19, 20 20, 21DH27 1, 2, 7, 13, 14, 19, 20 20, 21 DH25 1, 2, 6, 9, 13, 14, 19, 20 20,21 DH47 1, 2, 13, 14, 19, 20, 21 20, 21 DH29 1, 2, 13, 14, 18, 20, 2120, 21 DH28 1, 2, 13, 14, 18, 20, 21 None DH18 1, 2, 13, 14, 18, 20, 2119, 20, 21 DH36 1, 2, 20, 21 20, 21 DH9 1, 2, 13, 14, 20 20, 21 DH46 1,2, 13, 14, 21 20, 21 DH33 1, 2, 13, 14, 20, 21 20 DH10 1, 2, 13, 14, 2020 DH7 1, 2, 20, 21 2, 20, 21 DH23 1, 2, 13, 14 2, 20, 21 DH1 1, 2, 13,14, 20, 21 2, 20, 21 DH48 1, 2, 13, 14, 20, 21 2, 20 DH49 1, 2, 13, 14,20, 21 2, 21 DH44 1, 2, 13, 14, 20 2, 20, 21 DH45 1, 2, 13, 14, 21 2,20, 21 DH38 1, 2, 13, 14, 20, 21 1, 20, 21 DH39 1, 2, 13, 14, 20, 21 3,20, 21 DH40 1, 2, 13, 14, 20, 21 4, 20, 21 DH41 1, 2, 13, 14, 20, 21 5,20, 21 DH42 1, 2, 13, 14, 20, 21 6, 20, 21 DH43 1, 2, 13, 14, 20, 21 7,20, 21

Modified nucleotides introduced into the modification-formatted siRNAswere locked nucleic acid (LNA® residues), in particular those havingstructure (a) above where R1 is O, R2 is O, and R3 is CH₂.

The stability assay, as used herein, provides the amount of full-lengthproduct present after incubation of modification-formatted siRNA in 90%mouse serum that had not been heat inactivated (cat#44135, JRScientific, Inc., Woodland, Calif.). All siRNAs were treated with theidentical lot of mouse serum that had been validated as having robustnuclease activity prior to use. Similar results were obtained usinghuman serum (cat# CC-5500, SeraCare Life Sciences, Inc., Oceanside,Calif.) and fetal bovine serum (cat# SV30014, HyClone, Logan, Utah)(data not shown).

For each assay, 10 μM of modification-formatted siRNA in 50 μL finalvolume was incubated at 37° C. in 90% mouse serum for either the timeindicated in the time course (FIG. 17) or for a period of 5 hours (FIG.18A-FIG. 23B). Parallel to the incubation in serum, each set of siRNAswas treated with PBS (cat#9625, Applied Biosystems, Austin, Tex.) forthe same period of time as the serum-treated samples to establish abaseline for the amount of full-length product for HPLC studies.Following incubation, the siRNAs were extracted with phenol (cat#9700,Applied Biosystems, Austin, Tex.) and ethanol precipitated in thepresence of 5 μg glycogen carrier (1 μl/tube, Catalog No. AM9510,Applied Biosystems, Austin, Tex.) to enhance recovery of small RNAs.SiRNA and cleavage products were recovered by centrifugation (15 min at15,000×g) and dissolved in PBS buffer (phosphate-buffered saline) priorto HPLC analysis.

Determination of the amount of full-length product was carried out usingion-exchange high performance liquid chromatography (HPLC) using acontinuous gradient of sodium perchlorate (NaClO₄) containingacetonitrile. The stationary phase was a Dionex DNAPAC® 200 column (4mm×250 mm, Dionex Corporation, Sunnyvale, Calif.) used with a Waters2795 ALLIANCE® analytical HPLC system and EMPOWER™ Dissolution Softwarev2 (Waters Corp, Milford, Mass.). The temperature was maintained suchthat the siRNA duplex would not denature. Quantitation and UV detectionis performed at 254 nm via the Waters 2998 photodiode array detector(Waters Corp, Milford, Mass.). A siRNA (21-mer duplex with twooverhanging 3′ nucleotides on each end) targeting the mRNA for GAPdH (10μM) in a 10 μl-20 μl injection volume is used to ascertain columnquality and retention times. Analysis of the HPLC results was based onthe area counts of the peaks for untreated siRNA taken as 100%, and thearea counts of the peaks with the same retention time (+/−0.1-0.2 min)for the serum-treated samples providing a calculated remainingpercentage.

The same siRNAs tested for serum stability were tested separately forknockdown activity of endogenous genes to determine if the modificationformats altered the efficacy of the siRNA to knock down its mRNA target.Modification-formatted siRNAs and unmodified control siRNAs weretransfected into Hela cells at a final concentration of 5 nM asdescribed in Example 4 with the exception that the transfection usedLipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.) in the “forward”protocol described by the manufacturer. After harvesting transfectedcells, total cell lysate was isolated using the TaqMan® Gene ExpressionCells-to-CT™ kit (Applied Biosystems, Inc. Cat. #AM1728) according tothe manufacturer's protocol. Preparation of cDNA, gene expressionassays, and calculation of knockdown activity were carried out as inExample 4.

Degradation in serum of unmodified forms of the 8 different siRNAs ofthe present study is demonstrated by the data of FIG. 17 where, within 5to 10 min of exposure to serum, greater than 50% of the full lengthmolecule is degraded.

A box plot of the percentage of siRNAs that remain full length whentreated with 90% blood serum for 5 hours under described conditions isprovided by FIG. 18A for unmodified Format A, stability control FormatF, and modification formats H, DH21, DH20, DH3, DH30, DH35, DH6, DH34,and DH2. Unmodified Format A and modification Format H were completelydegraded in serum under the conditions described. Stability referencecontrol Format F has a median value of about 43% full length siRNA inserum under the conditions described. Analysis of the data of FIG. 18Aindicates modification of nucleotides at and/or near only one of the3′-termini is insufficient to protect the modified siRNA in serum(Formats DH21, DH20, DH3 and DH30 as compared to Format F). Modificationof two or three of the overhanging nucleotides is also insufficient toprotect the modified siRNA in serum (Formats DH6, DH34 and DH35 ascompared to Format F). Format DH2 having all four overhangingnucleotides modified provide protection in serum equal to or better thanFormat F. FIG. 18B provides a box plot of mRNA knockdown as a % of Negcontrol for the siRNAs studied for FIG. 18A. As shown by FIG. 18B,unmodified Format A siRNAs display a median knockdown of ˜85% comparedto Neg control siRNA treated samples. SiRNA modification Format DH20particularly shows a decrease in knockdown activity as compared to thatof Format H. Of this set of modification formats studied, only FormatDH2 is considered for further analysis due to its stability andknockdown activity.

Providing two modified nucleotides in the penultimate andantepenultimate positions of the 3′-terminus of the sense strandtogether with modified nucleotides in two or three of the terminalresidues of the antisense strands (DH4, DH31, DH19) appears to have adetrimental effect on serum stability as exhibited by FIG. 19A. Thesedata further support positioning modified nucleotides as the overhangingnucleotides. Moreover, as shown by the data of FIG. 19B, Formats DH19,DH27 and DH25 show a significant decrease in knockdown activity comparedto Format A. Format DH31 possessed knockdown activity of >70%.Protection of internal residues (as for DH27 (position 7) and DH25(positions 6 and 9)) while leaving terminal residues unprotected appearscounterproductive with regard to knockdown ability when the data arecompared to that of Format DH2.

The data provided by FIG. 20A demonstrate an increase in stability formodification Formats DH47 and DH29 and an even further increase instability for DH18 as compared to control Format F. Modification FormatsDH47 and DH29 provide for modified nucleotides at one of theantepenultimate and preantepenultimate positions of the sense strandsequence (i.e., for a 21-mer, one of positions 18 and 19) in addition tomodified nucleotides in the overhanging positions and positions ofFormat H. Format DH18 provides for modified nucleotides as for DH29 and,in addition, provides a modified nucleotide at the antepenultimateposition of the antisense strand (i.e., for a 21-mer, position 19). Thedata for Format DH28, when compared directly to Format DH29,demonstrates again that lack of modified nucleotides at the overhangingpositions of the antisense strand knocks out the stability provided bythe protected sense strand. When compared to Format F, data for FormatDH47 and DH28 have a Wilcoxon value where p<0.05. The data of FIG. 20Bdemonstrate that Formats DH47, DH 29, and DH28 possess knockdownactivity of >70%. Modification of the antepenultimate position of theantisense strand (position 19) as in Format DH18, while providingenhanced stability, compromises knockdown activity to a median of about60%. Based on the above data, Formats DH47 and DH29 both provideincreased stability of siRNA over control Format F, Format DH47 providesknockdown activity comparable to that of unmodified Format A and FormatDH29 provides knockdown activity of greater than 70%.

None of the siRNAs having modification formats depicted in FIG. 21Aprovide a serum stability increase over siRNAs having modificationFormat F or Format DH2. The lack of added serum stability furthersupports a modification format where the overhanging nucleotides aremodified. The knockdown activity data for the siRNAs studied for FIG.21A all demonstrate >70% activity as shown by FIG. 21B.

A comparison of serum stability of siRNAs having modification Format Fto that of siRNAs having modification Formats DH23, DH48, DH49, DH44,and DH45 of FIG. 22A further supports modified nucleotides at positionsof the overhanging nucleotides. Modification of the nucleotide atposition 2 of the antisense strand, in addition to having modifiednucleotides in the positions of the overhanging nucleotides, such as forFormat DH1 and DH7 increases the median for stability in serum (FIG.22A). Data of FIG. 22B demonstrate that all formatted siRNAs of FIG.22A, with the exception of Format DH23, achieve greater than 70%knockdown activity. Taken together, Formats DH1 and DH7, having amodified nucleotide at position 2 of the guide strand in addition topositions of overhanging nucleotides, provide both properties of serumstability and knockdown activity. SiRNAs having modification Format DH7lack modified nucleotides in the positions at and near 13 and 14 and,therefore, may not provide the property of removing off-target effects.However, such siRNAs may be useful for silencing in circumstances whereoff-target effects are not expected.

The data provided by FIG. 23A examine the effect of a one nucleotidemodification at positions 1, 2, 3, 4, 5, 6, or 7 of the antisense strand(position #1 is the 5′ nucleotide) in addition to modified nucleotidesof Format H and at the overhanging positions (DH38, DH1, DH39, DH40,DH41, DH42, DH43, respectively). The data demonstrate that median serumstability is increased by all of the formatted siRNAs tested with atrend to decreasing stability as the modified nucleotide is positionedfurther away from the 5′ end of the guide antisense strand.

As provided by the data of FIG. 23B, all formatted siRNAs have knockdownactivity of greater than 70% with the exception of siRNAs having FormatDH38. This observation highlights the incompatibility of a modifiednucleotide positioned at the 5′-end of the antisense strand. Addition ofa phosphate group to the 5′ end of the antisense strand partiallyrescued biological activity, but at the cost of reduced stability (datanot shown). Taken together, the data on serum stability and knockdowndemonstrate that an at least one nucleotide modification at positions 2,3, 4, 5, 6, or 7 of the guide antisense strand (with reference to the5′-end) enhances siRNA stability while also providing knockdown activityof at least 70%.

Taking the data of FIG. 18A-FIG. 23B together, those modificationformats that provide serum stability greater than Format F whilemaintaining knockdown activity of at least 70% are Formats DH47, DH29,DH7, DH1, DH39, DH40, DH41, DH42 and DH43.

Format DH7 lacks modified nucleotides in the positions at and nearpositions 13 and 14 and, therefore, may not provide the property ofremoving off-target effects.

Formats DH47, DH29, DH1, DH39, DH40, DH41, DH42 and DH43 have modifiednucleotides in the passenger sense strand at positions 1, 2, 13, 14, 20,21, and, for DH47 and DH29 one of positions 18 or 19; and have modifiednucleotides in the guide antisense strand at positions 20 and 21, and,for DH39-DH43 any one of positions 2, 3, 4, 5, 6, or 7. A duplexed basepair where both of the nucleotides are modified at position 1 of thepassenger sense strand appears detrimental to knockdown activity (DH20,DH19, DH4, DH18).

Example 8 In Vivo Delivery of Stabilized siRNAs

Unmodified Format A siRNA and stabilized siRNAs having modificationFormats DH1 and DH47 were delivered in vivo, and the amounts offull-length siRNA present in livers from control and test animals werecompared. The TaqMan® based stem-loop RT-PCR assay that allows reliableand robust quantification of siRNAs and miRNAs and specifically detectsonly full-length molecules was used for analysis (U.S. Published PatentApplication 2005/0266418 to Chen et al., filed Sep. 21, 2004; Chen, C.et al. Nucleic Acids Research 33, e179, 2005).

SiRNAs targeting the Fas gene were administered to mice (1 nmol dilutedin 2.5 ml PBS (10% of mouse body weight)) using hydrodynamic(high-pressure) tail vein injection as described by Zhang et al. (HumanGene Therapy 10, 1735-1737. 1999) and Lewis et al. (Nature Genetics 32,107-108. 2002). Four mice were injected for each modification-formattedsiRNA. The modified nucleotides were LNA® residues having structure (a)where R1 is O, R2 is O, and R3 is CH₂.

Five minutes post-injection, the mice were sacrificed and whole liverswere harvested and frozen in dry ice. Total RNA was isolated from wholelivers using the mirVana™ PARIS™ Kit (Applied Biosystems, Foster City,Calif.) according to the manufacturer's protocol with modifications asfollows. Cell disruption buffer (20 ml) was added and the samples werehomogenized. Lysates (400 μl) were transferred to new tubes, denaturingsolution (400 μl) was added to each tube and the tubes were mixed wellby vortexing. Phenol extractions were carried out by adding 800 μlphenol, vortexing for 5 min, and centrifuging for 10 min. The upperphase (300 μl) was transferred into new tubes and mixed with 375 μl(1.25 vol) 100% ethanol. The mixture was passed through filter columnsaccording to the protocol for the kit with a final elution of 100 μlelution buffer. RNA concentrations were measured and adjusted to 10ng/μl.

The assays for quantification of siRNAs included reverse transcription(RT) of the guide strand of the siRNA using the RT primer having astem-loop design as described for the TaqMan® MicroRNA ReverseTranscription Assays (Applied Biosystems, Foster City, Calif.) followedby PCR. In a 10 μl RT reaction, 1 ng/μl of total RNA and 50 nM of RTprimer were denatured at 85° C. for 5 min, then 60° C. for 5 min, andthen annealed at 4° C. After adding the enzyme mix (0.25 mM each of thedNTPs, final concentration; 3.33 units/μl of MultiScribe™ reversetranscriptase (Applied Biosystems), 1×RT buffer, 0.25 units/μl of RNaseinhibitor), the reaction mixture was incubated at 16° C. for 30 min, 42°C. for 30 min, 85° C. for 5 min, and then 4° C. Real-time PCR wasperformed using a standard TaqMan® PCR protocol on an Applied Biosystems7900HT Sequence Detection System (Applied Biosystems). The 10 μl PCRreaction mixture included 1 μl RT product, 1× TaqMan® Universal PCRMaster Mix, 0.2 μM TaqMan® probe, 1.5 μM forward primer, and 0.7 μMreverse primer. The reaction was incubated at 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min.

Frozen whole livers from a separate control untreated group of mice werespiked, prior to homogenization, with 300, 200, 100 and 50 pmol ofsiRNA. These “spiked” samples serve as a control for the study andprovide a standard curve for comparison of cycle threshold values (Ct)between the injected and the control samples. RNA was isolated from thecontrol organs as described above. The term “Ct” represents the PCRcycle number when the signal is first recorded as statisticallysignificant. Thus, the lower the Ct value, the greater the concentrationof nucleic acid target. In the TAQMAN® assay, typically each cyclealmost doubles the amount of PCR product and therefore, the fluorescentsignal should double if there is no inhibition of the reaction and thereaction was nearly 100% efficient with purified nucleic acid.

FIG. 24 provides the Ct values for the “spiked” controls, the unmodifiedFormat A injected animals, and the Format DH1 and Format DH47siRNA-treated animals. About 5% of the unmodified Format A siRNA wasdetected in the liver 5 minutes after hydrodynamic injection. Incontrast, about 20% of the modification-formatted siRNA having FormatsDH1 and DH47 were detected, a 400% increase compared to unmodifiedsiRNA. This in vivo study demonstrates that siRNAs that aremodification-formatted for stabilization provide for significantlyincreased delivery of full-length siRNA upon systemic administration.

The compositions, methods, and kits of the current teachings have beendescribed broadly and generically herein. Each of the narrower speciesand sub-generic groupings falling within the generic disclosure alsoform part of the current teachings. This includes the genericdescription of the current teachings with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications can be made without departingfrom the teachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings can befurther understood in light of the following claims.

The invention claimed is:
 1. A RNA, comprising: a passengeroligonucleotide having a length of 15 to 30 nucleotides and consistingof a modification format wherein nucleotides 1, 2, 13, and 14 aremodified wherein nucleotides are numbered from the 5′ end, and a guideoligonucleotide having a length of 15 to 30 nucleotides, a region ofcontinuous complementarity to the passenger oligonucleotide of at least12 nucleotides, and complementarity to at least a portion of targetmRNA, wherein each modified nucleotide is independently abicyclonucleotide, a tricyclonucleotide, or a 2′-modified nucleotide. 2.The RNA of claim 1 wherein the length of the passenger oligonucleotideis 18-25 nucleotides.
 3. The RNA of claim 1 wherein the length of thepassenger oligonucleotide is 19-22 nucleotides.
 4. The RNA of claim 1wherein the length of the passenger oligonucleotide is 19 nucleotides.5. The RNA of claim 1 wherein each modified nucleotide has structure(a):

wherein R1 and R2 are independently O, S, CH₂, or NR where R is hydrogenor C₁₋₃-alkyl; R3 is CH₂, CH₂—O, CH₂—S, CH₂—CH₂, CH₂—CH₂—CH₂, CH═CH, orCH₂—NR where R is hydrogen or C₁₋₃-alkyl; R4 and R5 are independently aninternucleoside linkage, a terminal group, or a protecting group; atleast one of R4 and R5 is an internucleoside linkage; and B is anucleobase, nucleobase derivative, or nucleobase analog.
 6. The RNA ofclaim 1 wherein at least one internucleoside linkage is other than aphosphodiester internucleoside linkage.
 7. The RNA of claim 1 whereinthe RNA is further associated with a cell-targeting ligand.
 8. The RNAof claim 1 wherein a 5′-end is further derivatized with a phosphate,C₁₋₁₂-alkyl, C₁₋₁₂-alkylamine, C₁₋₁₂-alkenyl, C₁₋₁₂-alkynyl,C₁₋₁₂-cycloalkyl, C₁₋₁₂-aralkyl, aryl, acyl, or silyl substituent.
 9. Acomposition comprising the RNA of claim 1 and a biologically acceptablecarrier.
 10. A kit comprising the RNA of claim 1 and a transfectionagent.
 11. A method of reducing off-target events for inhibition ofexpression of a target gene by RNA interference in a subject in needthereof, comprising: contacting the subject with the RNA of claim 1 inan amount sufficient to reduce off-target events while maintainingpotency.
 12. A method comprising: obtaining a RNA of claim 1 whereineach modified nucleotide has structure (a):

wherein R1 and R2 are independently O, S, CH₂, or NR where R is hydrogenor C₁₋₃-alkyl; R3 is CH₂, CH₂—O, CH₂—S, CH₂—CH₂, CH₂—CH₂—CH₂, CH═CH, orCH₂—NR where R is hydrogen or C₁₋₃-alkyl; R4 and R5 are independently aninternucleoside linkage, a terminal group, or a protecting group; atleast one of R4 and R5 is an internucleoside linkage; B is a nucleobase,nucleobase derivative, or nucleobase analog; and administering said RNAin vivo to a subject in need thereof; wherein, post administration, agreater amount of said RNA is present in vivo as compared to an amountof RNA lacking modified nucleotides.
 13. The method of claim 11 whereinthe gene is an endogenous gene.
 14. The method of claim 11 wherein thecontacting is in vitro contacting of a cell culture containing the cell,or a tissue containing the cell with the RNA.
 15. The method of claim 11wherein the contacting is ex vivo contacting of a tissue containing thecell, a bodily fluid containing the cell, or an organ containing thecell with the RNA.
 16. The method of claim 11 wherein the contacting isin vivo contacting of an organ or an animal containing the cell with theRNA.
 17. The RNA of claim 1 wherein the 2′ modification is OR wherein Ris alkyl and wherein the alkyl is C1 to C6.
 18. The RNA of claim 17wherein the alkyl is methyl.